Platelet derived growth factor (PDGF) nucleic acid ligand complexes

ABSTRACT

This invention discloses a method for preparing a complex comprised of a PDGF Nucleic Acid Ligand and a Non-Immunogenic, High Molecular Weight Compound or Lipophilic Compound by identifying a PDGF Nucleic Acid Ligand by SELEX methodology and associating the PDGF Nucleic Acid Ligand with a Non-Immunogenic, High Molecular Weight Compound or Lipophilic Compound. The invention further discloses Complexes comprising one or more PDGF Nucleic Acid Ligands in association with a Non-Immunogenic, High Molecular Weight Compound or Lipophilic Compound. The invention further includes a Lipid construct comprising a PDGF Nucleic Acid Ligand or Complex and methods for making the same.

RELATED APPLICATIONS

This application is a Continuation-in-Part of U.S. patent applicationSer. No. 08/618,693, filed Mar. 20, 1996, now U.S. Pat. No. 5,723,594which is a Continuation-in-Part of U.S. patent application Ser. No.08/479,783, filed Jun. 7, 1995, now U.S. Pat. No. 5,668,264, and U.S.Ser. No. 08/479,725, filed Jun. 7, 1995 issued as U.S. Pat. No.5,674,685.

FIELD OF THE INVENTION

Described herein are high affinity ssDNA and RNA ligands to plateletderived growth factor (PDGF). The method utilized herein for identifyingsuch Nucleic Acid Ligands is called SELEX, an acronym for SystematicEvolution of Ligands by Exponential enrichment. Further included in thisinvention is a method for preparing a therapeutic or diagnostic Complexcomprised of a PDGF Nucleic Acid Ligand and a Non-Immunogenic, HighMolecular Weight Compound or a Lipophilic Compound by identifying a PDGFNucleic Acid Ligand by SELEX methodology and covalently linking the PDGFNucleic Acid Ligand with a Non-Immunogenic, High Molecular WeightCompound or a Lipophilic Compound. The invention further includesComplexes comprised of one or more PDGF Nucleic Acid Ligands and aNon-Immunogenic, High Molecular Weight Compound or a LipophilicCompound. The invention further relates to improving the PharmacokineticProperties of a PDGF Nucleic Acid Ligand by covalently linking the PDGFNucleic Acid Ligand with a Non-Immunogenic, High Molecular WeightCompound or Lipophilic Compound to form a Complex. The invention furtherrelates to improving the Pharmacokinetic Properties of a PDGF NucleicAcid Ligand by using a Lipid Construct comprising a PDGF Nucleic AcidLigand or a Complex comprising a PDGF Nucleic Acid Ligand and aNon-Immunogenic, High Molecular Weight Compound or Lipophilic Compound.This invention further relates to a method for targeting a therapeuticor diagnostic agent to a biological target that is expressing PDGF byassociating the agent with a Complex comprised of a PDGF Nucleic AcidLigand and a Lipophilic Compound or Non-Immunogenic, High MolecularWeight Compound, wherein the Complex is further associated with a LipidConstruct and the PDGF Nucleic Acid Ligand is further associated withthe exterior of the Lipid Construct.

BACKGROUND OF THE INVENTION

A. The SELEX Process

The dogma for many years was that nucleic acids had primarily aninformational role. Through a method known as Systematic Evolution ofLigands by Exponential enrichment, termed SELEX, it has become clearthat nucleic acids have three dimensional structural diversity notunlike proteins. SELEX is a method for the in vitro evolution of nucleicacid molecules with highly specific binding to target molecules and isdescribed in U.S. patent application Ser. No. 07/536,428, filed Jun. 11,1990, entitled “Systematic Evolution of Ligands by ExponentialEnrichment,” now abandoned, U.S. patent application Ser. No. 07/714,131,filed Jun. 10, 1991, entitled “Nucleic Acid Ligands,” now U.S. Pat. No.5,475,096, and U.S. patent application Ser. No. 07/931,473, filed Aug.17, 1992, entitled “Methods of Identifying Nucleic Acid Ligands,” nowU.S. Pat. No. 5,270,163 (see also WO 91/19813), each of which isspecifically incorporated by reference herein. Each of theseapplications, collectively referred to herein as the SELEX patentApplications, describes a fundamentally novel method for making aNucleic Acid Ligand to any desired target molecule. The SELEX processprovides a class of products which are referred to as Nucleic AcidLigands, each ligand having a unique sequence, and which has theproperty of binding specifically to a desired target compound ormolecule. Each SELEX-identified Nucleic Acid Ligand is a specific ligandof a given target compound or molecule. SELEX is based on the uniqueinsight that Nucleic Acids have sufficient capacity for forming avariety of two- and three-dimensional structures and sufficient chemicalversatility available within their monomers to act as ligands (formspecific binding pairs) with virtually any chemical compound, whethermonomeric or polymeric. Molecules of any size or composition can serveas targets.

The SELEX method involves selection from a mixture of candidateoligonucleotides and step-wise iterations of binding, partitioning andamplification, using the same general selection scheme, to achievevirtually any desired criterion of binding affinity and selectivity.Starting from a mixture of Nucleic Acids, preferably comprising asegment of randomized sequence, the SELEX method includes steps ofcontacting the mixture with the target under conditions favorable forbinding, partitioning unbound Nucleic Acids from those Nucleic Acidswhich have bound specifically to target molecules, dissociating theNucleic Acid-target complexes, amplifying the Nucleic Acids dissociatedfrom the Nucleic Acid-target complexes to yield a ligand-enrichedmixture of Nucleic Acids, then reiterating the steps of binding,partitioning, dissociating and amplifying through as many cycles asdesired to yield highly specific high affinity Nucleic Acid Ligands tothe target molecule.

It has been recognized by the present inventors that the SELEX methoddemonstrates that Nucleic Acids as chemical compounds can form a widearray of shapes, sizes and configurations, and are capable of a farbroader repertoire of binding and other functions than those displayedby Nucleic Acids in biological systems.

The present inventors have recognized that SELEX or SELEX-like processescould be used to identify Nucleic Acids which can facilitate any chosenreaction in a manner similar to that in which Nucleic Acid Ligands canbe identified for any given target. In theory, within a CandidateMixture of approximately 10¹³ to 10¹⁸ Nucleic Acids, the presentinventors postulate that at least one Nucleic Acid exists with theappropriate shape to facilitate each of a broad variety of physical andchemical interactions.

The basic SELEX method has been modified to achieve a number of specificobjectives. For example, U.S. patent application Ser. No. 07/960,093,filed Oct. 14, 1992, entitled “Method for Selecting Nucleic Acids on theBasis of Structure,” describes the use of SELEX in conjunction with gelelectrophoresis to select Nucleic Acid molecules with specificstructural characteristics, such as bent DNA. U.S. patent applicationSer. No. 08/123,935, filed Sep. 17, 1993, entitled “Photoselection ofNucleic Acid Ligands,” describes a SELEX based method for selectingNucleic Acid Ligands containing photoreactive groups capable of bindingand/or photocrosslinking to and/or photoinactivating a target molecule.U.S. patent application Ser. No. 08/134,028, filed Oct. 7, 1993,entitled “High-Affinity Nucleic Acid Ligands That Discriminate BetweenTheophylline and Caffeine,” now U.S. Pat. No. 5,580,737, describes amethod for identifying highly specific Nucleic Acid Ligands able todiscriminate between closely related molecules, which can benon-peptidic, termed Counter-SELEX. U.S. patent application Ser. No.08/143,564, filed Oct. 25, 1993, entitled “Systematic Evolution ofLigands by Exponential Enrichment: Solution SELEX,” now U.S. Pat. No.5,567,588, describes a SELEX-based method which achieves highlyefficient partitioning between oligonucleotides having high and lowaffinity for a target molecule.

The SELEX method encompasses the identification of high-affinity NucleicAcid Ligands containing modified nucleotides conferring improvedcharacteristics on the ligand, such as improved in vivo stability orimproved delivery characteristics. Examples of such modificationsinclude chemical substitutions at the ribose and/or phosphate and/orbase positions. SELEX-identified Nucleic Acid Ligands containingmodified nucleotides are described in U.S. patent application Ser. No.08/117,991, filed Sep. 8, 1993, entitled “High Affinity Nucleic AcidLigands Containing Modified Nucleotides,” now U.S. Pat. No. 5,660,985,that describes oligonucleotides containing nucleotide derivativeschemically modified at the 5- and 2′-positions of pyrimidines. U.S.patent application Ser. No. 08/134,028, supra, describes highly specificNucleic Acid Ligands containing one or more nucleotides modified with2′-amino (2′-NH₂), 2′-fluoro (2′-F), and/or 2′-O-methyl (2′-OMe). U.S.patent application Ser. No. 08/264,029, filed Jun. 22, 1994, entitled“Novel Method of Preparation of Known and Novel 2′Modified Nucleosidesby Intramolecular Nucleophilic Displacement,” describes oligonucleotidescontaining various 2′-modified pyrimidines.

The SELEX method encompasses combining selected oligonucleotides withother selected oligonucleotides and non-oligonucleotide functional unitsas described in U.S. patent application Ser. No. 08/284,063, filed Aug.2, 1994, entitled “Systematic Evolution of Ligands by ExponentialEnrichment: Chimeric SELEX,” now U.S. Pat. No. 5,637,459, and U.S.patent application Ser. No. 08/234,997, filed Apr. 28, 1994, entitled“Systematic Evolution of Ligands by Exponential Enrichment: BlendedSELEX,” now U.S. Pat. No. 5,683,867, respectively. These applicationsallow the combination of the broad array of shapes and other properties,and the efficient amplification and replication properties, ofoligonucleotides with the desirable properties of other molecules.

The SELEX method further encompasses combining selected nucleic acidligands with lipophilic compounds or non-immunogenic, high molecularweight compounds in a diagnostic or therapeutic complex as described inU.S. patent application Ser. No. 08/434,465, filed May 4, 1995, entitled“Nucleic Acid Ligand Complexes,” now U.S. Pat No. 6,011,020. VEGFNucleic Acid Ligands that are associated with a Lipophilic Compound,such as diacyl glycerol or dialkyl glycerol, in a diagnostic ortherapeutic complex are described in U.S. patent application Ser. No.08/739,109, filed Oct. 25, 1996, entitled “Vascular Endothelial GrowthFactor (VEGF) Nucleic Acid Ligand Complexes,” now U.S. Pat. No.5,859,228. VEGF Nucleic Acid Ligands that are associated with aLipophilic Compound, such as a glycerol lipid, or a Non-Immunogenic,High Molecular Weight Compound, such as polyethylene glycol, are furtherdescribed in U.S. patent application Ser. No. 08/897,351, filed Jul. 21,1997, entitled “Vascular Endothelial Growth Factor (VEGF) Nucleic AcidLigand Complexes,” now U.S. Pat. No. 6,051,698. VEGF Nucleic AcidLigands that are associated with a non-immunogenic, high molecularweight compound or lipophilic compound are also further described inPCT/US97/18944, filed Oct. 17, 1997, entitled “Vascular EndothelialGrowth Factor (VEGF) Nucleic Acid Ligand Complexes.” Each of the abovedescribed patent applications which describe modifications of the basicSELEX procedure are specifically incorporated by reference herein intheir entirety.

B. LIPID CONSTRUCTS

Lipid Bilayer Vesicles are closed, fluid-filled microscopic sphereswhich are formed principally from individual molecules having polar(hydrophilic) and non-polar (lipophilic) portions. The hydrophilicportions may comprise phosphate, glycerylphosphato, carboxy, sulfato,amino, hydroxy, choline or other polar groups. Examples of lipophilicgroups are saturated or unsaturated hydrocarbons such as alkyl, alkenylor other lipid groups. Sterols (e.g., cholesterol) and otherpharmaceutically acceptable adjuvants (including anti-oxidants likealpha-tocopherol) may also be included to improve vesicle stability orconfer other desirable characteristics.

Liposomes are a subset of these bilayer vesicles and are comprisedprincipally of phospholipid molecules that contain two hydrophobic tailsconsisting of fatty acid chains. Upon exposure to water, these moleculesspontaneously align to form spherical, bilayer membranes with thelipophilic ends of the molecules in each layer associated in the centerof the membrane and the opposing polar ends forming the respective innerand outer surface of the bilayer membrane(s). Thus, each side of themembrane presents a hydrophilic surface while the interior of themembrane comprises a lipophilic medium. These membranes may be arrangedin a series of concentric, spherical membranes separated by thin strataof water, in a manner not dissimilar to the layers of an onion, aroundan internal aqueous space. These multilamellar vesicles (MLV) can beconverted into small or Unilamellar Vesicles (UV), with the applicationof a shearing force.

The therapeutic use of liposomes includes the delivery of drugs whichare normally toxic in the free form. In the liposomal form, the toxicdrug is occluded, and may be directed away from the tissues sensitive tothe drug and targeted to selected areas. Liposomes can also be usedtherapeutically to release drugs over a prolonged period of time,reducing the frequency of administration. In addition, liposomes canprovide a method for forming aqueous dispersions of hydrophobic oramphiphilic drugs, which are normally unsuitable for intravenousdelivery.

In order for many drugs and imaging agents to have therapeutic ordiagnostic potential, it is necessary for them to be delivered to theproper location in the body, and the liposome can thus be readilyinjected and form the basis for sustained release and drug delivery tospecific cell types, or parts of the body. Several techniques can beemployed to use liposomes to target encapsulated drugs to selected hosttissues, and away from sensitive tissues. These techniques includemanipulating the size of the liposomes, their net surface charge, andtheir route of administration. MLVs, primarily because they arerelatively large, are usually rapidly taken up by thereticuloendothelial system (principally the liver and spleen). UVs, onthe other hand, have been found to exhibit increased circulation times,decreased clearance rates and greater biodistribution relative to MLVs.

Passive delivery of liposomes involves the use of various routes ofadministration, e.g., intravenous, subcutaneous, intramuscular andtopical. Each route produces differences in localization of theliposomes. Two common methods used to direct liposomes actively toselected target areas involve attachment of either antibodies orspecific receptor ligands to the surface of the liposomes. Antibodiesare known to have a high specificity for their corresponding antigen andhave been attached to the surface of liposomes, but the results havebeen less than successful in many instances. Some efforts, however, havebeen successful in targeting liposomes to tumors without the use ofantibodies, see, for example, U.S. Pat. No. 5,019,369, U.S. Pat. No.5,441,745, or U.S. Pat. No. 5,435,989.

An area of development aggressively pursued by researchers is thedelivery of agents not only to a specific cell type but into the cell'scytoplasm and, further yet, into the nucleus. This is particularlyimportant for the delivery of biological agents such as DNA, RNA,ribozymes and proteins. A promising therapeutic pursuit in this areainvolves the use of antisense DNA and RNA oligonucleotides for thetreatment of disease. However, one major problem encountered in theeffective application of antisense technology is that oligonucleotidesin their phosphodiester form are quickly degraded in body fluids and byintracellular and extracellular enzymes, such as endonucleases andexonucleases, before the target cell is reached. Intravenousadministration also results in rapid clearance from the bloodstream bythe kidney, and uptake is insufficient to produce an effectiveintracellular drug concentration. Liposome encapsulation protects theoligonucleotides from the degradative enzymes, increases the circulationhalf-life and increases uptake efficiency as a result of phagocytosis ofthe Liposomes. In this way, oligonucleotides are able to reach theirdesired target and to be delivered to cells in vivo.

A few instances have been reported where researchers have attachedantisense oligonucleotides to Lipophilic Compounds or Non-Immunogenic,High Molecular Weight Compounds. Antisense oligonucleotides, however,are only effective as intracellular agents. Antisenseoligodeoxyribonucleotides targeted to the epidermal growth factor (EGF)receptor have been encapsulated into Liposomes linked to folate via apolyethylene glycol spacer (folate-PEG-Liposomes) and delivered intocultured KB cells via folate receptor-mediated endocytosis (Wang et al.(1995) Proc. Natl. Acad. Sci. USA 92:3318-3322). In addition, aklylenediols have been attached to oligonucleotides (Weiss et al., U.S. Pat.No. 5,245,022). Furthermore, a Lipophilic Compound covalently attachedto an antisense oligonucleotide has been demonstrated in the literature(EP 462 145 B1).

Loading of biological agents into liposomes can be accomplished byinclusion in the lipid formulation or loading into preformed liposomes.Passive anchoring of oligopeptide and oligosaccharide ligands to theexternal surface of liposomes has been described (Zalipsky etal. (1997)Bioconjug. Chem. 8:111:118).

C. PDGF

Platelet-derived growth factor (PDGF) was originally isolated fromplatelet lysates and identified as the major growth-promoting activitypresent in serum but not in plasma. Two homologous PDGF isoforms havebeen identified, PDGF A and B, which are encoded by separate genes (onchromosomes 7 and 22). The most abundant species from platelets is theAB heterodimer, although all three possible dimers (AA, AB and BB) occurnaturally. Following translation, PDGF dimers are processed into ≈30 kDasecreted proteins. Two cell surface proteins that bind PDGF with highaffinity have been identified, α and β (Heldin et al. (1981) Proc. Natl.Acad. Sci., 78: 3664; Williams et al. (1981) Proc. Natl. Acad. Sci., 79:5867). Both species contain five immunoglobulin-like extracellulardomains, a single transmembrane domain and an intracellular tyrosinekinase domain separated by a kinase insert domain. The functional highaffinity receptor is a dimer and engagement of the extracellular domainof the receptor by PDGF results in cross-phosphorylation (one receptortyrosine kinase phosphorylates the other in the dimer) of severaltyrosine residues. Receptor phosphorylation leads to a cascade of eventsthat results in the transduction of the mitogenic or chemotactic signalto the nucleus. For example, in the intracellular domain of the PDGF Breceptor, nine tyrosine residues have been identified that whenphosphorylated interact with different src-homology 2 (SH2)domain-containing proteins including phospholipase C-g,phosphatidylinositol 3′-kinase, GTPase-activating protein and severaladapter molecules like Shc, Grb2 and Nck (Heldin (1995) Cell 80: 213).In the last several years, the specificities of the three PDGF isoformsfor the three receptor dimers (αα, αβ, and ββ) has been elucidated. Theα-receptor homodimer binds all three PDGF isoforms with high affinity,the β-receptor homodimer binds only PDGF BB with high affinity and PDGFAB with approximately 10-fold lower affinity, and the αβ-receptorheterodimer binds PDGF BB and PDGF AB with high affinity (Westermark &Heldin (1993) Acta Oncologica 32:101). The specificity pattern resultsfrom the ability of the A-chain to bind only to the α-receptor and ofthe B-chain to bind to both a and β-receptor subunits with highaffinity.

The role of PDGF in proliferative diseases, such as cancer, restenosis,fibrosis, angiogenesis, and wound healing has been established.

PDGF in Cancer

The earliest indication that PDGF expression is linked to malignanttransformation came with the finding that the amino acid sequence ofPDGF-B chain is virtually identical to that of p28^(sis), thetransforming protein of the simian sarcoma virus (SSV) (Waterfield etal. (1983) Nature 304:35; Johnson et al. (1984) EMBO J. 3:921). Thetransforming potential of the PDGF-B chain gene and, to a lesser extent,the PDGF-A gene was demonstrated soon thereafter (Clarke et al. (1984)Nature 308:464; Gazit et al. (1984) Cell 39:89; Beckmann et al. Science241:1346; Bywater et al. (1988) Mol. Cell. Biol. 8:2753). Many tumorcell lines have since been shown to produce and secrete PDGF, some ofwhich also express PDGF receptors (Raines et al. (1990) Peptide GrowthFactors and Their Receptors, Springer-Verlag, Part I, p 173). Paracrineand, in some cell lines, autocrine growth stimulation by PDGF istherefore possible. For example, analysis of biopsies from human gliomashas revealed the existence of two autocrine loops: PDGF-B/β-receptor intumor-associated endothelial cells and PDGF-A/α-receptor in tumor cells(Hermansson et al. (1988) Proc. Natl. Acad. Sci. 85:7748; Hermansson etal. (1992) Cancer Res. 52:3213). The progression to high grade gliomawas accompanied by the increase in expression of PDGF-B and theβ-receptor in tumor-associated endothelial cells and PDGF-A in gliomacells. PDGF overexpression may thus promote tumor growth either bydirectly stimulating tumor cells or by stimulating tumor-associatedstromal cells (e.g., endothelial cells). The proliferation ofendothelial cells is a hallmark of angiogenesis. Increased expression ofPDGF and/or PDGF receptors has also been observed in other malignanciesincluding fibrosarcoma (Smits et al. (1992) Am. J. Pathol. 140:639) andthyroid carcinoma (Heldin et al. (1991) Endocrinology 129:2187).

PDGF in Cardiovascular Disease

Percutaneous transluminal coronary angioplasty (PTCA) has become themost common treatment for occlusive coronary artery disease (CAD)involving one or two coronary arteries. In the United States alone about500,000 procedures are being done annually, with projections of over700,000 procedures by the year 2000 and about double those amountsworldwide. PTCA, while it involves manipulations inside of coronaryarteries, is not considered to be a cardiac surgical intervention.During the most common PTCA procedure, a balloon catheter is threadedthrough a femoral artery and is positioned within the plaque-ladensegment of an occluded coronary vessel; once in place, the balloon isexpanded at high pressure, compressing the plaque and increasing thevessel lumen. Unfortunately, in 30-50% of PTCA procedures, reocclusiongradually develops over a period of several weeks or months due tocellular events in the affected vessel wall. Once reocclusion achieves50% or greater reduction of the original vessel lumen, clinicalrestenosis is established in the vessel.

In view of the increasing popularity of coronary angioplasty as a lessinvasive alternative to bypass surgery, restenosis is a serious medicalproblem. Smooth muscle cells (SMCs) represent a major component of therestenosis lesions. In uninjured arteries, SMCs reside primarily in themedial vessel layer (tunical media). Upon balloon injury that removesthe endothelial cells from the intimal layer (tunical intima), SMCsproliferate and migrate into the intima, forming neointimal thickeningcharacteristic of restenosis lesions. When restenosis occurs subsequentto angioplasty, it is usually treated by repeat angioplasty, with orwithout placement of a stent, or by vascular graft surgery (bypass).

A stent is a rigid cylindrical mesh that, once placed and expandedwithin a diseased vessel segment, mechanically retains the expandedvessel wall. The stent is deployed by catheter and, having beenpositioned at the desired site, is expanded in situ by inflation of ahigh pressure balloon. Being rigid and non-compressible, the expandedstent achieves and maintains a vessel lumen diameter comparable to thatof adjacent non-diseased vessel; being pressed tightly into theoverlying intima/media, it is resistant to migration within the vesselin response to blood flow. PTCA with stent placement has been comparedwith PTCA alone and shown to reduce restenosis to about half and tosignificantly improve other clinical outcomes such as myocardialinfarction (MI) and need for bypass surgery.

There is now considerable evidence that PDGF B-chain is a majorcontributor to the formation of neointimal lesions. In a rat model ofrestenosis, the neointimal thickening was inhibited with anti-PDGF-Bantibodies (Ferns (1991) Science 253:1129-1132; Rutherford et al. (1997)Atherosclerosis 130:45-51). Conversely, the exogenous administration ofPDGF-BB promotes SMC migration and causes an increase in neointimalthickening (Jawien et al. (1992) J. Clin. Invest. 89:507-511). Theeffect of PDGF-B on SMCs is mediated through PDGF β-receptor which isexpressed at high levels in these cells after balloon injury (Lindnerand Reidy (1995) Circulation Res. 76:951-957). Furthermore, the degreeof neointimal thickening following balloon injury was found to beinversely related to the level of expression of PDGF β-receptor at thesite of injury (Sirois et al. (1997) Circulation 95:669-676).

U.S. Pat. No. 5,171,217 discloses a method and composition for deliveryof a drug to an affected intramural site for sustained release inconjunction with or following balloon catheter procedures, such asangioplasty. The drug may be selected from a variety of drugs known toinhibit smooth muscle cell proliferation, including growth factorreceptor antagonists for PDGF.

U.S. Pat. No. 5,593,974 discloses methods for treating vasculardisorders, such as vascular restenosis, with antisense oligonucleotides.The method is based on localized application of the antisenseoligonucleotides to a specific site in vivo. The oligonucleotides can beapplied directly to the target tissue in a mixture with an implant orgel, or by direct injection or infusion.

U.S. Pat. No. 5,562,922 discloses a method for preparing a systemsuitable for localized delivery of biologically active compounds to asubject. The method relates to treating polyurethane coated substratewith a coating expansion solution under conditions that will allowpenetration of the biologically active compound throughout thepolyurethane coating. Substrates suitable for this invention include,intel, metallic stents. Biologically active compounds suitable for usein this invention include, inter alia, lipid-modified oligonucleotides.

Rutherford et al. (1997, Atherosclerosis 130:45-51) report substantialinhibition of neointimal response to balloon injury in rat carotidartery using a combination of antibodies to PDGF-BB and basic fibroblastgrowth factor (bFGF).

PDGF in Renal Disease

A large variety of progressive renal diseases are characterized byglomerular mesangial cell proliferation and matrix accumulation(Slomowitz et al. (1988) New Eng. J. Med. 319:1547-1548) which leads tofibrosis. PDGF B-chain appears to have a central role in driving both ofthese processes given that 1) mesangial cells produce PDGF in vitro andvarious growth factors induce mesangial proliferation via induction ofauto- or paracrine PDGF B-chain synthesis; 2) PDGF B-chain and itsreceptor are overexpressed in many glomerular diseases; 3) infusion ofPDGF-BB or glomerular transfection with a PDGF B-chain cDNA can induceselective mesangial cell proliferation and matrix accumulation in vivo;and 4) PDGF B-chain or βreceptor knock-out mice fail to develop amesangium (reviewed in Floege and Johnson (1995) Miner. ElectrolyteMetab. 21:271-282). In addition to contributing to kidney fibrosis, PDGFis also believed to play a role in fibrosis development in other organssuch as lungs and bone marrow and may have other possible diseaseassociations (Raines et al. (1990) Experimental Pharmacology, PeptideGrowth Factors and Their Receptors, Spom & Roberts, eds., pp. 173-262,Springer, Heidelberg).

One study has examined the effect of inhibition of PDGF B-chain in renaldisease: Johnson et al., using a neutralizing polyclonal antibody toPDGF, were able to reduce mesangial cell proliferation and matrixaccumulation in experimental mesangioproliferative glomerulonephritis(Johnson et al. (1992) J. Exp. Med. 175:1413-1416). In this model,injection of an anti-mesangial cell antibody (anti-Thy 1.1) into ratsresulted in complement-dependent lysis of the mesangial cells, followedby an overshooting reparative phase that resembled humanmesangioproliferative nephritis (Floege et al. (1993) Kidney Int. Suppl.39: S47-54). Limitations of the study of Johnson et al. (Johnson et al.(1992) J. Exp. Med. 175:1413-1416) included the necessity to administerlarge amounts of heterologous IgG and a limitation of the study durationto 4 days due to concerns that the heterologous IgG might elicit animmune reaction.

Inhibition of PDGF

Specific inhibition of growth factors, such as PDGF, has become a majorgoal in experimental and clinical medicine. However, this approach isusually hampered by the lack of specific pharmacological antagonists.Available alternative approaches are also limited, since neutralizingantibodies often show a low efficacy in vivo and are usuallyimmunogenic, and given that in vivo gene therapy for these purposes isstill in its infancy. Currently, antibodies to PDGF (Johnson et al.(1985) Proc. Natl. Acad. Sci. U.S.A. 82:1721-1725; Ferns et al. (1991)Science 253:1129-1132; Herren et al. (1993) Biochimica et BiophysicaActa 1173:294-302; Rutherford et al. (1997) Atherosclerosis 130:45-51)and the soluble PDGF receptors (Herren et al. (1993) Biochimica etBiophysica Acta 1173:294-302; Duan et al. (1991) J. Biol. Chem.266:413-418; Teisman et al. (1993) J. Biol. Chem. 268:9621-9628) are themost potent and specific antagonists of PDGF. Neutralizing antibodies toPDGF have been shown to revert the SSV-transformed phenotype (Johnssonet al. (1985) Proc. Natl. Acad. Sci. U.S.A. 82:1721-1725) and to inhibitthe development of neointimal lesions following arterial injury (Fernset al. (1991) Science 253:1129-1132). Other inhibitors of PDGF such assuramin (Williams et al. (1984) J. Biol. Chem. 259:287-5294; Betsholtzet al. (1984) Cell 39:447-457), neomycin (Vassbotn et al. (1992) J.Biol. Chem. 267:15635-15641) and peptides derived from the PDGF aminoacid sequence (Engström et al. (1992) J. Biol. Chem. 267:16581-16587)have been reported, however, they are either too toxic or lacksufficient specificity or potency to be good drug candidates. Othertypes of antagonists of possible clinical utility are molecules thatselectively inhibit the PDGF receptor tyrosine kinase (Buchdunger et al.(1995) Proc. Natl. Acad. Sci. U.S.A. 92:2558-2562; Kovalenko et al.(1994) Cancer Res. 54:6106-6114).

SUMMARY OF THE INVENTION

The present invention includes methods of identifying and producingnucleic acid ligands to platelet-derived growth factor (PDGF) andhomologous proteins and the nucleic acid ligands so identified andproduced. For the purposes of this application, PDGF refers to PDGF AA,AB, and BB isoforms and homologous proteins. Specifically included inthe definition are human PDGF AA, AB, and BB isoforms.

Described herein are high affinity ssDNA and RNA ligands to plateletderived growth factor (PDGF). The method utilized herein for identifyingsuch nucleic acid ligands is called SELEX, an acronym for SystematicEvolution of Ligands by Exponential enrichment. Included herein are theevolved ligands that are shown in Tables 2-3, 6-7, and 9 and FIGS. 1-2,8A, 8B and 9A. Further included in this invention is a method forpreparing a Complex comprised of a PDGF Nucleic Acid Ligand and aNon-Immunogenic, High Molecular Weight Compound or Lipophilic Compoundby the method comprising identifying a Nucleic Acid Ligand from aCandidate Mixture of Nucleic Acids where the Nucleic Acid is a ligand ofPDGF by the method of (a) contacting the Candidate Mixture of NucleicAcids with PDGF, (b) partitioning between members of said CandidateMixture on the basis of affinity to PDGF, and c) amplifying the selectedmolecules to yield a mixture of Nucleic Acids enriched for Nucleic Acidsequences with a relatively higher affinity for binding to PDGF, andcovalently linking said identified PDGF Nucleic Acid Ligand with aNon-Immunogenic, High Molecular Weight Compound or a LipophilicCompound. The invention further comprises a Complex comprised of a PDGFNucleic Acid Ligand and a Non-Immunogenic, High Molecular WeightCompound or a Lipophilic Compound.

The invention further includes a Lipid Construct comprising a PDGFNucleic Acid Ligand or a Complex. The present invention further relatesto a method for preparing a Lipid Construct comprising a Complex whereinthe Complex is comprised of a PDGF Nucleic Acid Ligand and a LipophilicCompound.

In another embodiment, this invention provides a method for improvingthe pharmacokinetic properties of a PDGF Nucleic Acid Ligand bycovalently linking the PDGF Nucleic Acid Ligand with a Non-Immunogenic,High Molecular Weight Compound or Lipophilic Compound to form a Complexand administering the Complex to a patient. The invention furtherrelates to a method for improving the pharmacokinetic properties of aPDGF Nucleic Acid Ligand by further associating the Complex with a LipidConstruct.

It is an object of the present invention to provide Complexes comprisingone or more PDGF Nucleic Acid Ligands in association with one or moreNon-Immunogenic, High Molecular Weight Compounds or Lipophilic Compoundsand methods for producing the same. It is a further object of thepresent invention to provide Lipid Constructs comprising a Complex. Itis a further object of the invention to provide one or more PDGF NucleicAcid Ligands in association with one or more Non-Immunogenic, HighMolecular Weight Compounds or Lipophilic Compounds with improvedPharmacokinetic Properties.

In embodiments of the invention directed to Complexes comprised of aPDGF Nucleic Acid Ligand and a Non-Immunogenic, High Molecular WeightCompound, it is preferred that the Non-Immunogenic, High MolecularWeight Compound is Polyalkylene Glycol, more preferably, polyethyleneglycol (PEG). More preferably, the PEG has a molecular weight of about10-80K. Most preferably, the PEG has a molecular weight of about 20-45K.In embodiments of the invention directed to Complexes comprised of aPDGF Nucleic Acid Ligand and a Lipophilic Compound, it is preferred thatthe Lipophilic Compound is a glycerolipid. In the preferred embodimentsof the invention, the Lipid Construct is preferably a Lipid BilayerVesicle and most preferably a Liposome. In the preferred embodiment, thePDGF Nucleic Acid Ligand is identified according to the SELEX method.

In embodiments of the invention directed to Complexes comprising aNon-Immunogenic, High Molecular Weight Compound or Lipophilic Compoundcovalently linked to a PDGF Nucleic Acid Ligand or Ligands, the PDGFNucleic Acid Ligand or Ligands can serve in a targeting capacity.

Additionally, the PDGF Nucleic Acid Ligand can be associated throughCovalent or Non-Covalent Interactions with a Lipid Construct withoutbeing part of a Complex.

Furthermore, in embodiments of the invention directed to LipidConstructs comprising a PDGF Nucleic Acid Ligand or a Non-Immunogenic,High Molecular Weight or Lipophilic Compound/PDGF Nucleic Acid LigandComplex where the Lipid Construct is of a type that has a membranedefining an interior compartment such as a Lipid Bilayer Vesicle, thePDGF Nucleic Acid Ligand or Complex in association with the LipidConstruct may be associated with the membrane of the Lipid Construct orencapsulated within the compartment. In embodiments where the PDGFNucleic Acid Ligand is in association with the membrane, the PDGFNucleic Acid Ligand can associate with the interior-facing orexterior-facing part of the membrane, such that the PDGF Nucleic AcidLigand is projecting into or out of the vesicle. In certain embodiments,a PDGF Nucleic Acid Ligand Complex can be passively loaded onto theoutside of a preformed Lipid Construct. In embodiments where the NucleicAcid Ligand is projecting out of the Lipid Construct, the PDGF NucleicAcid Ligand can serve in a targeting capacity.

In embodiments where the PDGF Nucleic Acid Ligand of the Lipid Constructserves in a targeting capacity, the Lipid Construct can have associatedwith it additional therapeutic or diagnostic agents. In one embodiment,the therapeutic or diagnostic agent is associated with the exterior ofthe Lipid Construct. In other embodiments, the therapeutic or diagnosticagent is encapsulated in the Lipid Construct or associated with theinterior of the Lipid Construct. In yet a further embodiment, thetherapeutic or diagnostic agent is associated with the Complex. In oneembodiment, the therapeutic agent is a drug. In an alternativeembodiment, the therapeutic or diagnostic agent is one or moreadditional Nucleic Acid Ligands.

It is a further object of the present invention to provide a method forinhibiting PDGF-mediated diseases. PDGF-mediated diseases include, butare not limited to, cancer, angiogenesis, restenosis, and fibrosis.Thus, it is a further object of the present invention to provide amethod for inhibiting angiogenesis by the administration of a PDGFNucleic Acid Ligand or a Complex comprising a PDGF Nucleic Acid Ligandand Non-Immunogenic, High Molecular Weight Compound or LipophilicCompound or a Lipid Construct comprising the Complex of the presentinvention. It is yet a further object of the present invention toprovide a method for inhibiting the growth of tumors by theadministration of a PDGF Nucleic Acid Ligand or Complex comprising aPDGF Nucleic Acid Ligand and Non-Immunogenic, High Molecular WeightCompound or Lipophilic Compound or a Lipid Construct comprising aComplex of the present invention. It is yet a further object of theinvention to provide a method for inhibiting fibrosis by theadministration of a PDGF Nucleic Acid Ligand or Complex comprising aPDGF Nucleic Acid Ligand and Non-Immunogenic, High Molecular WeightCompound or Lipophilic Compound or a Lipid Construct comprising aComplex of the present invention. It is yet a further object of theinvention to provide a method for inhibiting restenosis by theadministration of a PDGF Nucleic Acid Ligand or Complex comprising aPDGF Nucleic Acid Ligand and Non-Immunogenic, High Molecular WeightCompound or Lipophilic Compound or a Lipid Construct comprising aComplex of the present invention

It is a further object of the invention to provide a method fortargeting a therapeutic or diagnostic agent to a biological target thatis expressing PDGF by associating the agent with a Complex comprised ofa PDGF Nucleic Acid Ligand and a Lipophilic Compound or Non-Immunogenic,High Molecular Weight Compound, wherein the Complex is furtherassociated with a Lipid Construct and the PDGF Nucleic Acid Ligand isfurther associated with the exterior of the Lipid Construct.

These and other objects, as well as the nature, scope and utilization ofthis invention, will become readily apparent to those skilled in the artfrom the following description and the appended claims.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the consensus secondary structure for the sequence setshown in Table 3. R=A or G, Y=C or T, K=G or T, N and N′ indicate anybase pair.

FIG. 2 shows the minimal ligands 20t, 36t and 41t folded according tothe consensus secondary structure motif. [3′T] represents a 3′-3′ linkedthymidine nucleotide added to reduce 3′-exonuclease degradation.

FIGS. 3A-3C show the binding of minimal high affinity DNA ligands toPDGF AA, PDGF AB, and PDGF BB, respectively. The fraction of ³²P 5′end-labeled DNA ligands bound to varying concentrations of PDGF wasdetermined by the nitrocellulose filter binding method. Minimal ligandstested were 20t (∘), 36t (Δ), and 41t (□). Oligonucleotideconcentrations in these experiments were ≈10 pM (PDGF-AB and PDGF-BB)and ≈50 pM (PDGF AA). Data points were fitted to eq. 1 (for binding ofthe DNA ligands to PDGF-AA) or to eq. 2 (for binding to PDGF AB and BB)using the non-linear least squares method. Binding reactions were doneat 37° C. in binding buffer (PBSM with 0.01% HSA).

FIG. 4 shows the dissociation rate determination for the high affinityinteraction between the minimal DNA ligands and PDGF AB. The fraction of5′ ³²P end-labeled ligands 20t (∘), 36t (Δ), and 41t (□), all at 0.17nM, bound to PDGF AB (1 nM) was measured by nitrocellulose filterbinding at the indicated time points following the addition of a500-fold excess of the unlabeled competitor. The dissociation rateconstant (k_(off)) values were determined by fitting the data points toeq 3 in Example 1. The experiments were performed at 37° C. in bindingbuffer.

FIG. 5 shows the thermal denaturation profiles for the minimal highaffinity DNA ligands to PDGF-AB. The change in absorbance at 260 nm wasmeasured in PBS containing 1 mM MgCl₂ as a function of temperature forligands 20t (∘), 36t (Δ), and 41t (□).

FIG. 6 shows the effect of DNA ligands on the binding of ¹²⁵I-PDGF-BB toPDGF α-receptors expressed in PAE cells.

FIG. 7 shows the effect of DNA ligands on the mitogenic effect ofPDGF-BB on PAE cells expressing the PDGF β-receptors.

FIGS. 8A-8B show the substitution pattern compatible with high affinitybinding to PDGF-AB. In FIGS. 8A-8C, the underlined symbols indicate2′-O-methyl-2′-deoxynucleotides; italicized symbols indicate2′-fluoro-2′-deoxynucleotides; normal font indicates2′-deoxyribonucleotides; [3′T] indicates inverted orientation (3′3′)thymidine nucleotide (Glen Research, Sterling, Va.); PEG in the loops ofhelices II and III of FIG. 8B indicates pentaethylene glycol spacerphosphoramidite (Glen Research, Sterling, Va.) (See FIG. 9 for moleculardescription). FIG. 8C shows the predicted secondary structure of ascrambled Nucleic Acid Ligand sequence that was used as a control inExamples 8 and 9. The scrambled region is boxed to accent the overallsimilarity of the scrambled Nucleic Acid Ligand to the Nucleic AcidLigand shown in FIG. 8B.

FIGS. 9A-9E show the molecular descriptions NX31975 40K PEG (FIG. 9A),NX31976 40K(FIG. 9B), hexaethylene glycol phosphoramidite (FIG. 9C),pentyl amino linker (FIG. 9D), and 40K PEG NHS ester (FIG. 9E). The 5′phosphate group shown in the PEG Spacer of FIGS. 9A and 9B are from thehexaethylene glycol phosphoramidite.

FIG. 10 shows the stabilities of DNA (36ta) and modified DNA (NX21568)Nucleic Acid Ligands in rat serum over time at 37° C. were compared.36ta is shown by the symbol ▪; and NX21568 is shown by the symbol ▴.

FIG. 11 shows that NX31975-40K PEG significantly inhibited (p<0.05)about 50% of the neointima formation in rats based on the intima/mediaratio for the control (PBS) and NX31975-40K PEG groups.

FIG. 12 shows the effects of NX31975 40K PEG on mitogen-stimulatedproliferation of mesangial cells in culture (all mitogens were added at100 ng/ml final concentration). Scrambled Nucleic Acid Ligand NX31976and 40K PEG were also tested. Data are optical densities measured in theXTT assay and are expressed as percentages of baseline, i.e., cellsstimulated with medium plus 200 μg/ml 40K PEG (i.e., the amountequivalent to the PEG attached to 50 μg/ml Nucleic Acid Ligand). Resultsare means±SD of 5 separate experiments (n=3 in the case of medium plus40K PEG; statistical evaluation was therefore confined to NX31975 andscrambled Nucleic Acid Ligand groups).

FIGS. 13A-13E show effects of NX31975 40K PEG on glomerular cellproliferation (FIG. 13A), expression of glomerular PDGF B-chain (FIG.13B), proteinuria in rats with anti-Thy 1.1 nephritis (FIG. 13C),mesangial cell activation (as assessed by glomerular de novo expressionof α-smooth muscle actin) (FIG. 13D), and monocyte/macrophage influx(FIG. 13E). NX31975 40K PEG is shown as black, NX31976 40K PEG is shownas cross-hatched, 40K PEG is shown as white, PBS is shown as hatched,and the normal range is shown as stippled.

FIGS. 14A-C shows the effects of NX31975 40K PEG on glomerular matrixaccumulation. Glomerular immunostaining scores for fibronectin and typeIV collagen as well as glomerular scores for type IV collagen mRNAexpression (in situ hydridization) are shown. NX31975 40K PEG is shownas black, NX31976 40K PEG is shown as cross-hatched, 40K PEG is shown aswhite, PBS is shown as hatched, and the normal range is shown asstippled.

DETAILED DESCRIPTION OF THE INVENTION

DEFINITIONS:

“Covalent Bond” is the chemical bond formed by the sharing of electrons.

“Non-Covalent Interactions” are means by which molecular entities areheld together by interactions other than Covalent Bonds including ionicinteractions and hydrogen bonds.

“Lipophilic Compounds” are compounds which have the propensity toassociate with or partition into lipid and/or other materials or phaseswith low dielectric constants, including structures that are comprisedsubstantially of lipophilic components. Lipophilic Compounds includelipids as well as non-lipid containing compounds that have thepropensity to associate with lipid (and/or other materials or phaseswith low dielectric constants). Cholesterol, phospholipids, andglycerolipids, such as dialkylglycerol, and diacylglycerol, and glycerolamide lipids are further examples of Lipophilic Compounds.

“Complex” as used herein describes the molecular entity formed by thecovalent linking of a PDGF Nucleic Acid Ligand to a Non-Immunogenic,High Molecular Weight Compound or Lipophilic Compound. In certainembodiments of the present invention, the Complex is depicted as A-B-Y,wherein A is a Lipophilic Compound or Non-Immunogenic, High MolecularWeight Compound as described herein; B is optional, and comprises aSpacer which may comprise one or more linkers Z; and Y is a PDGF NucleicAcid Ligand.

“Lipid Constructs,” for purposes of this invention, are structurescontaining lipids, phospholipids, or derivatives thereof comprising avariety of different structural arrangements which lipids are known toadopt in aqueous suspension. These structures include, but are notlimited to, Lipid Bilayer Vesicles, micelles, Liposomes, emulsions,lipid ribbons or sheets, and may be complexed with a variety of drugsand components which are known to be pharmaceutically acceptable. In thepreferred embodiment, the Lipid Construct is a Liposome. The preferredLiposome is unilamellar and has a relative size less than 200 nm. Commonadditional components in Lipid Constructs include cholesterol andalpha-tocopherol, among others. The Lipid Constructs may be used aloneor in any combination which one skilled in the art would appreciate toprovide the characteristics desired for a particular application. Inaddition, the technical aspects of Lipid Constructs and Liposomeformation are well known in the art and any of the methods commonlypracticed in the field may be used for the present invention.

“Nucleic Acid Ligand” as used herein is a non-naturally occurringNucleic Acid having a desirable action on a Target. The Target of thepresent invention is PDGF, hence the term PDGF Nucleic Acid Ligand. Adesirable action includes, but is not limited to, binding of the Target,catalytically changing the Target, reacting with the Target in a waywhich modifies/alters the Target or the functional activity of theTarget, covalently attaching to the Target as in a suicide inhibitor,facilitating the reaction between the Target and another molecule. Inthe preferred embodiment, the action is specific binding affinity forPDGF, wherein the Nucleic Acid Ligand is not a Nucleic Acid having theknown physiological function of being bound by PDGF.

In preferred embodiments of the invention, the PDGF Nucleic Acid Ligandof the Complexes and Lipid Constructs of the invention are identified bythe SELEX methodology. PDGF Nucleic Acid Ligands are identified from aCandidate Mixture of Nucleic Acids, said Nucleic Acid being a ligand ofPDGF, by the method comprising a) contacting the Candidate Mixture withPDGF, wherein Nucleic Acids having an increased affinity to PDGFrelative to the Candidate Mixture may be partitioned from the remainderof the Candidate Mixture; b) partitioning the increased affinity NucleicAcids from the remainder of the Candidate Mixture; and c) amplifying theincreased affinity Nucleic Acids to yield a ligand-enriched mixture ofNucleic Acids (see U.S. patent application Ser. No. 08/479,725, filedJun. 7, 1995, entitled “High Affinity PDGF Nucleic Acid Ligands,” nowU.S. Pat. No. 5,674,685, U.S. patent application Ser. No. 08/479,783,filed Jun. 7, 1995, entitled “High Affinity PDGF Nucleic Acid Ligands,”now U.S. Pat. No. 5,668,264, and U.S. patent application Ser. No.08/618,693, filed Mar. 20, 1996, entitled “High Affinity PDGF NucleicAcid Ligands,” now U.S. Pat. No. 5,723,594, which are herebyincorporated by reference herein).

In certain embodiments, portions of the PDGF Nucleic Acid Ligand (Y) maynot be necessary to maintain binding and certain portions of thecontiguous PDGF Nucleic Acid Ligand can be replaced with a Spacer orLinker. In these embodiments, for example, Y can be represented asY-B′-Y′-B″-Y″, wherein Y, Y′ and Y″ are parts of a PDGF Nucleic AcidLigand or segments of different PDGF Nucleic Acid Ligands and B′ and/orB″ are Spacers or Linker molecules that replace certain nucleic acidfeatures of the original PDGF Nucleic Acid Ligand. When B′ and B″ arepresent and Y, Y′, and Y″ are parts of one PDGF Nucleic Acid Ligand, atertiary structure is formed that binds to PDGF. When B′ and B″ are notpresent, Y, Y′, and Y″ represent one contiguous PDGF Nucleic AcidLigand. PDGF Nucleic Acid Ligands modified in such a manner are includedin this definition.

“Candidate Mixture” is a mixture of Nucleic Acids of differing sequencefrom which to select a desired ligand. The source of a Candidate Mixturecan be from naturally-occurring Nucleic Acids or fragments thereof,chemically synthesized Nucleic Acids, enzymatically synthesized NucleicAcids or Nucleic Acids made by a combination of the foregoingtechniques. In a preferred embodiment, each Nucleic Acid has fixedsequences surrounding a randomized region to facilitate theamplification process.

“Nucleic Acid” means either DNA, RNA, single-stranded or double-strandedand any chemical modifications thereof Modifications include, but arenot limited to, those which provide other chemical groups thatincorporate additional charge, polarizability, hydrogen bonding,electrostatic interaction, and fluxionality to the Nucleic Acid Ligandbases or to the Nucleic Acid Ligand as a whole. Such modificationsinclude, but are not limited to, 2′-position sugar modifications,5-position pyrimidine modifications, 8-position purine modifications,modifications at exocyclic amines, substitution of 4-thiouridine,substitution of 5-bromo or 5-iodo-uracil, backbone modifications such asintemucleoside phosphorothioate linkages, methylations, unusualbase-pairing combinations such as the isobases isocytidine andisoguanidine and the like. Modifications can also include 3′ and 5′modifications such as capping.

“Non-Immunogenic, High Molecular Weight Compound” is a compound betweenapproximately 1000 Da to 1,000,000 Da, more preferably approximately1000 Da to 500,000 Da, and most preferably approximately 1000 Da to200,000 Da, that typically does not generate an immunogenic response.For the purposes of this invention, an immunogenic response is one thatcauses the organism to make antibody proteins. Examples ofNon-Immunogenic, High Molecular Weight Compounds include PolyalkyleneGlycol and polyethylene glycol. In one preferred embodiment of theinvention, the Non-Immunogenic, High Molecular Weight Compoundcovalently linked to the PDGF Nucleic Acid Ligand is a polyalkyleneglycol and has the structure R(O(CH₂)_(x))_(n)O—, where R isindependently selected from the group consisting of H and CH₃, x=2-5,and n≈MW of the Polyalkylene Glycol/(16+14x). In the preferredembodiment of the present invention, the molecular weight is aboutbetween 10-80 kDa. In the most preferred embodiment, the molecularweight of the polyalkylene glycol is about between 20-45kDa. In the mostpreferred embodiment, x=2 and n=9×10². There can be one or morePolyalkylene Glycols attached to the same PDGF Nucleic Acid Ligand, withthe sum of the molecular weights preferably being between 10-80kDa, morepreferably 20-45 kDa.

In certain embodiments, the Non-Immunogenic, High Molecular WeightCompound can also be a Nucleic Acid Ligand.

“Lipid Bilayer Vesicles” are closed, fluid-filled microscopic sphereswhich are formed principally from individual molecules having polar(hydrophilic) and non-polar (lipophilic) portions. The hydrophilicportions may comprise phosphato, glycerylphosphato, carboxy, sulfato,amino, hydroxy, choline and other polar groups. Examples of non-polargroups are saturated or unsaturated hydrocarbons such as alkyl, alkenylor other lipid groups. Sterols (e.g., cholesterol) and otherpharmaceutically acceptable components (including anti-oxidants likealpha-tocopherol) may also be included to improve vesicle stability orconfer other desirable characteristics.

“Liposomes” are a subset of Lipid Bilayer Vesicles and are comprisedprincipally of phospholipid molecules which contain two hydrophobictails consisting of long fatty acid chains. Upon exposure to water,these molecules spontaneously align to form a bilayer membrane with thelipophilic ends of the molecules in each layer associated in the centerof the membrane and the opposing polar ends forming the respective innerand outer surface of the bilayer membrane. Thus, each side of themembrane presents a hydrophilic surface while the interior of themembrane comprises a lipophilic medium. These membranes when formed aregenerally arranged in a system of concentric closed membranes separatedby interlamellar aqueous phases, in a manner not dissimilar to thelayers of an onion, around an internal aqueous space. Thesemultilamellar vesicles (MLV) can be converted into unilamellar vesicles(UV), with the application of a shearing force.

“Cationic Liposome” is a Liposome that contains lipid components thathave an overall positive charge at physiological pH.

“SELEX” methodology involves the combination of selection of NucleicAcid Ligands which interact with a Target in a desirable manner, forexample binding to a protein, with amplification of those selectedNucleic Acids. Iterative cycling of the selection/amplification stepsallows selection of one or a small number of Nucleic Acids whichinteract most strongly with the Target from a pool which contains a verylarge number of Nucleic Acids. Cycling of the selection/amplificationprocedure is continued until a selected goal is achieved. The SELEXmethodology is described in the SELEX patent Applications.

“Target” means any compound or molecule of interest for which a ligandis desired. A Target can be a protein (such as PDGF, thrombin, andselectin), peptide, carbohydrate, polysaccharide, glycoprotein, hormone,receptor, antigen, antibody, virus, substrate, metabolite, transitionstate analog, cofactor, inhibitor, drug, dye, nutrient, growth factor,etc. without limitation. The principal Target of the subject inventionis PDGF.

“Improved Pharmacokinetic Properties” means that the PDGF Nucleic AcidLigand covalently linked to a Non-Immunogenic, High Molecular WeightCompound or Lipophilic Compound or in association with a Lipid Constructshows a longer circulation half-life in vivo relative to the same PDGFNucleic Acid Ligand not in association with a Non-Immunogenic, HighMolecular Weight Compound or Lipophilic Compound or in association witha Lipid Construct.

“Linker” is a molecular entity that connects two or more molecularentities through Covalent Bond or Non-Covalent Interactions, and canallow spatial separation of the molecular entities in a manner thatpreserves the functional properties of one or more of the molecularentities. A linker can also be known as a Spacer. Examples of Linkers,include but are not limited to, the structures shown in FIGS. 9C-9E andthe PEG spacer shown in FIG. 9A.

In the preferred embodiment, the linker B′ and B″ are pentaethyleneglycols.

“Therapeutic” as used herein, includes treatment and/or prophylaxis.When used, Therapeutic refers to humans and other animals.

This invention includes ssDNA and RNA ligands to PDGF. This inventionfurther includes the specific RNA ligands to PDGF shown in Tables 2-3,6-7, and 9 and FIGS. 1-2, 8A and 8B (SEQ ID NOS:4-35, 39-87, 97-149).More specifically, this invention includes nucleic acid sequences thatare substantially homologous to and that have substantially the sameability to bind PDGF as the specific nucleic acid ligands shown inTables 2-3, 6-7, and 9 and FIGS. 1-2, 8A and 8B (SEQ ID NOS:4-35, 39-87,97-149). By substantially homologous it is meant a degree of primarysequence homology in excess of 70%, most preferably in excess of 80%,and even more preferably in excess of 90%, 95%, or 99%. The percentageof homology as described herein is calculated as the percentage ofnucleotides found in the smaller of the two sequences which align withidentical nucleotide residues in the sequence being compared when 1 gapin a length of 10 nucleotides may be introduced to assist in thatalignment. Substantially the same ability to bind PDGF means that theaffinity is within one or two orders of magnitude of the affinity of theligands described herein. It is well within the skill of those ofordinary skill in the art to determine whether a givensequence—substantially homologous to those specifically describedherein—has the same ability to bind PDGF.

A review of the sequence homologies of the nucleic acid ligands of PDGFshown in Tables 2-3, 6-7, and 9 and FIGS. 1-2, 8A and 8B (SEQ IDNOS:4-35, 39-87, 97-149) shows that sequences with little or no primaryhomology may have substantially the same ability to bind PDGF. For thesereasons, this invention also includes Nucleic Acid Ligands that havesubstantially the same postulated structure or structural motifs andability to bind PDGF as the nucleic acid ligands shown in Tables 2-3,6-7, and 9 and FIGS. 1-2, 8A and 8B (SEQ ID NOS:4-35, 39-87, 97-149).Substantially the same structure or structural motifs can be postulatedby sequence alignment using the Zukerfold program (see Zuker (1989)Science 244:48-52). As would be known in the art, other computerprograms can be used for predicting secondary structure and structuralmotifs. Substantially the same structure or structural motif of NucleicAcid Ligands in solution or as a bound structure can also be postulatedusing NMR or other techniques as would be known in the art.

Further included in this invention is a method for preparing a Complexcomprised of a PDGF Nucleic Acid Ligand and a Non-Immunogenic, HighMolecular Weight Compound or Lipophilic Compound by the methodcomprising identifying a Nucleic Acid Ligand from a Candidate Mixture ofNucleic Acids where the Nucleic Acid is a ligand of PDGF by the methodof (a) contacting the Candidate Mixture of Nucleic Acids with PDGF, (b)partitioning between members of said Candidate Mixture on the basis ofaffinity to PDGF, and c) amplifying the selected molecules to yield amixture of Nucleic Acids enriched for Nucleic Acid sequences with arelatively higher affinity for binding to PDGF, and covalently linkingsaid identified PDGF Nucleic Acid Ligand with a Non-Immunogenic, HighMolecular Weight Compound or a Lipophilic Compound.

It is a further object of the present invention to provide Complexescomprising one or more PDGF Nucleic Acid Ligands covalently linked to aNon-Immunogenic, High Molecular Weight Compound or Lipophilic Compound.Such Complexes have one or more of the following advantages over a PDGFNucleic Acid Ligand not in association with a Non-Immunogenic, HighMolecular Weight Compound or Lipophilic Compound: 1) ImprovedPharmacokinetic Properties, and 2) improved capacity for intracellulardelivery, or 3) improved capacity for targeting. Complexes furtherassociated with a Lipid Construct have the same advantages.

The Complexes or the Lipid Constructs comprising the PDGF Nucleic AcidLigand or Complexes may benefit from one, two, or three of theseadvantages. For example, a Lipid Construct of the present invention maybe comprised of a) a Liposome, b) a drug that is encapsulated within theinterior of the Liposome, and c) a Complex comprised of a PDGF NucleicAcid Ligand and Lipophilic Compound, wherein the PDGF Nucleic AcidLigand component of the Complex is associated with and projecting fromthe exterior of the Lipid Construct. In such a case, the Lipid Constructcomprising a Complex will 1) have Improved Pharmacokinetic Properties,2) have enhanced capacity for intracellular delivery of the encapsulateddrug, and 3) be specifically targeted to the preselected location invivo that is expressing PDGF by the exteriorly associated PDGF NucleicAcid Ligand.

In another embodiment, this invention provides a method for improvingthe pharmacokinetic properties of a PDGF Nucleic Acid Ligand bycovalently linking the PDGF Nucleic Acid Ligand with a Non-Immunogenic,High Molecular Weight Compound or Lipophilic Compound to form a Complexand administering the Complex to a patient. The invention furtherrelates to a method for improving the pharmacokinetic properties of aPDGF Nucleic Acid Ligand by further associating the Complex with a LipidConstruct.

In another embodiment, the Complex of the present invention is comprisedof a PDGF Nucleic Acid Ligand covalently attached to a LipophilicCompound, such as a glycerolipid, or a Non-Immunogenic, High MolecularWeight Compound, such as Polyalkylene Glycol or polyethylene glycol(PEG). In these cases, the pharmacokinetic properties of the Complexwill be enhanced relative to the PDGF Nucleic Acid Ligand alone. Inanother embodiment, the pharmacokinetic properties of the PDGF NucleicAcid Ligand is enhanced relative to the PDGF Nucleic Acid Ligand alonewhen the PDGF Nucleic Acid Ligand is covalently attached to aNon-Immunogenic, High Molecular Weight Compound or Lipophilic Compoundand is further associated with a Lipid Construct or the PDGF NucleicAcid Ligand is encapsulated within a Lipid Construct.

In embodiments where there are multiple PDGF Nucleic Acid Ligands, thereis an increase in avidity due to multiple binding interactions withPDGF. Furthermore, in embodiments where the Complex is comprised ofmultiple PDGF Nucleic Acid Ligands, the pharmacokinetic properties ofthe Complex will be improved relative to one PDGF Nucleic Acid Ligandalone. In embodiments where a Lipid Construct comprises multiple NucleicAcid Ligands or Complexes, the Pharmacokinetic Properties of the PDGFNucleic Acid Ligand may be improved relative to Lipid Constructs inwhich there is only one Nucleic Acid Ligand or Complex.

In certain embodiments of the invention, the Complex of the presentinvention is comprised of a PDGF Nucleic Acid Ligand attached to one(dimeric) or more (multimeric) other Nucleic Acid Ligands. The NucleicAcid Ligand can be to PDGF or a different Target. In embodiments wherethere are multiple PDGF Nucleic Acid Ligands, there is an increase inavidity due to multiple binding interactions with PDGF. Furthermore, inembodiments of the invention where the Complex is comprised of a PDGFNucleic Acid Ligand attached to one or more other PDGF Nucleic AcidLigands, the pharmacokinetic properties of the Complex will be improvedrelative to one PDGF Nucleic Acid Ligand alone.

The Non-Immunogenic, High Molecular Weight compound or LipophilicCompound may be covalently bound to a variety of positions on the PDGFNucleic Acid Ligand, such as to an exocyclic amino group on the base,the 5-position of a pyrimidine nucleotide, the 8-position of a purinenucleotide, the hydroxyl group of the phosphate, or a hydroxyl group orother group at the 5′ or 3′ terminus of the PDGF Nucleic Acid Ligand. Inembodiments where the Non-Immunogenic, High Molecular Weight Compound ispolyalkylene glycol or polyethylene glycol, preferably it is bonded tothe 5′ or 3′ hydroxyl of the phosphate group thereof. In the mostpreferred embodiment, the Non-Immunogenic, High Molecular WeightCompound is bonded to the 5′ hydroxyl of the phosphate group of theNucleic Acid Ligand. Attachment of the Non-Immunogenic, High MolecularWeight Compound or Lipophilic Compound to the PDGF Nucleic Acid Ligandcan be done directly or with the utilization of Linkers or Spacers. Inembodiments where the Lipid Construct comprises a Complex, or where thePDGF Nucleic Acid Ligands are encapsulated within the Liposome, aNon-Covalent Interaction between the PDGF Nucleic Acid Ligand or theComplex and the Lipid Construct is preferred.

One problem encountered in the therapeutic use of Nucleic Acids is thatoligonucleotides in their phosphodiester form may be quickly degraded inbody fluids by intracellular and extracellular enzymes such asendonucleases and exonucleases before the desired effect is manifest.Certain chemical modifications of the PDGF Nucleic Acid Ligand can bemade to increase the in vivo stability of the PDGF Nucleic Acid Ligandor to enhance or to mediate the delivery of the PDGF Nucleic AcidLigand. Modifications of the PDGF Nucleic Acid Ligands contemplated inthis invention include, but are not limited to, those which provideother chemical groups that incorporate additional charge,polarizability, hydrophobicity, hydrogen bonding, electrostaticinteraction, and fluxionality to the PDGF Nucleic Acid Ligand bases orto the PDGF Nucleic Acid Ligand as a whole. Such modifications include,but are not limited to, 2′-position sugar modifications, 5-positionpyrimidine modifications, 8-position purine modifications, modificationsat exocyclic amines, substitution of 4-thiouridine, substitution of5-bromo or 5-iodo-uracil; backbone modifications, phosphorothioate oralkyl phosphate modifications, methylations, unusual base-pairingcombinations such as the isobases isocytidine and isoguanidine and thelike. Modifications can also include 3′ and 5′ modifications such ascapping.

Where the Nucleic Acid Ligands are derived by the SELEX method, themodifications can be pre- or post-SELEX modifications. Pre-SELEXmodifications yield PDGF Nucleic Acid Ligands with both specificity forPDGF and improved in vivo stability. Post-SELEX modifications made to2′-OH Nucleic Acid Ligands can result in improved in vivo stabilitywithout adversely affecting the binding capacity of the Nucleic AcidLigands. The preferred modifications of the PDGF Nucleic Acid Ligands ofthe subject invention are 5′ and 3′ phosphorothioate capping and 3′3′inverted phosphodiester linkage at the 3′ end. In the most preferredembodiment, the preferred modification of the PDGF Nucleic Acid Ligandis 3′3′ inverted phosphodiester linkage at the 3end. Additional 2′fluoro (2′-F), 2′ amino (2′-NH₂) and 2′ OMethyl (2′-OMe) modification ofall or some of the nucleotides is preferred. In the most preferredembodiment, the preferred modification is 2′-OMe and 2′-F modificationof some of the nucleotides. Additionally, the PDGF Nucleic Acid Ligandcan be post-SELEX modified to substitute Linkers or Spacers such ashexaethylene glycol Spacers for certain portions.

In another aspect of the present invention, the covalent linking of thePDGF Nucleic Acid Ligand with a Non-Immunogenic, High Molecular WeightCompound or Lipophilic Compound results in Improved PharmacokineticProperties (i.e., slower clearance rate) relative to the PDGF NucleicAcid Ligand not in association with a Non-Immunogenic, High MolecularWeight Compound or Lipophilic Compound.

In another aspect of the present invention, the Complex comprising aPDGF Nucleic Acid Ligand and Non-Immunogenic, High Molecular WeightCompound or Lipophilic Compound can be further associated with a LipidConstruct. This association may result in Improved PharmacokineticProperties relative to the PDGF Nucleic Acid Ligand or Complex not inassociation with a Lipid Construct. The PDGF Nucleic Acid Ligand orComplex can be associated with the Lipid Construct through covalent orNon-Covalent Interactions. In another aspect, the PDGF Nucleic AcidLigand can be associated with the Lipid Construct through Covalent orNon-Covalent Interactions. In a preferred embodiment, the association isthrough Non-Covalent Interactions. In a preferred embodiment, the LipidConstruct is a Lipid Bilayer Vesicle. In the most preferred embodiment,the Lipid Construct is a Liposome.

Liposomes for use in the present invention can be prepared by any of thevarious techniques presently known in the art or subsequently developed.Typically, they are prepared from a phospholipid, for example,distearoyl phosphatidylcholine, and may include other materials such asneutral lipids, for example, cholesterol, and also surface modifierssuch as positively charged (e.g., sterylamine or aminomannose oraminomannitol derivatives of cholesterol) or negatively charged (e.g.,diacetyl phosphate, phosphatidyl glycerol) compounds. MultilamellarLiposomes can be formed by conventional techniques, that is, bydepositing a selected lipid on the inside wall of a suitable containeror vessel by dissolving the lipid in an appropriate solvent, and thenevaporating the solvent to leave a thin film on the inside of the vesselor by spray drying. An aqueous phase is then added to the vessel with aswirling or vortexing motion which results in the formation of MLVs. UVscan then be formed by homogenization, sonication or extrusion (throughfilters) of MLV's. In addition, UVs can be formed by detergent removaltechniques.

In certain embodiments of this invention, the Lipid Construct comprisesa targeting PDGF Nucleic Acid Ligand(s) associated with the surface ofthe Lipid Construct and an encapsulated therapeutic or diagnostic agent.Preferably the Lipid Construct is a Liposome. Preformed Liposomes can bemodified to associate with the PDGF Nucleic Acid Ligands. For example, aCationic Liposome associates through electrostatic interactions with thePDGF Nucleic Acid Ligand. A PDGF Nucleic Acid Ligand covalently linkedto a Lipophilic Compound, such as a glycerolipid, can be added topreformed Liposomes whereby the glycerolipid, phospholipid, or glycerolamide lipid becomes associated with the liposomal membrane.Alternatively, the PDGF Nucleic Acid Ligand can be associated with theLiposome during the formulation of the Liposome.

It is well known in the art that Liposomes are advantageous forencapsulating or incorporating a wide variety of therapeutic anddiagnostic agents. Any variety of compounds can be enclosed in theinternal aqueous compartment of the Liposomes. Illustrative therapeuticagents include antibiotics, antiviral nucleosides, antifungalnucleosides, metabolic regulators, immune modulators, chemotherapeuticdrugs, toxin antidotes, DNA, RNA, antisense oligonucleotides, etc. Bythe same token, the Lipid Bilayer Vesicles may be loaded with adiagnostic radionuclide (e.g., Indium 111, Iodine 131, Yttrium 90,Phosphorous 32, or gadolinium) and fluorescent materials or othermaterials that are detectable in in vitro and in vivo applications. Itis to be understood that the therapeutic or diagnostic agent can beencapsulated by the Liposome walls in the aqueous interior.Alternatively, the carried agent can be a part of, that is, dispersed ordissolved in the vesicle wall-forming materials.

During Liposome formation, water soluble carrier agents may beencapsulated in the aqueous interior by including them in the hydratingsolution, and lipophilic molecules incorporated into the lipid bilayerby inclusion in the lipid formulation. In the case of certain molecules(e.g., cationic or anionic lipophilic drugs), loading of the drug intopreformed Liposomes may be accomplished, for example, by the methodsdescribed in U.S. Pat. No. 4,946,683, the disclosure of which isincorporated herein by reference. Following drug encapsulation, theLiposomes are processed to remove unencapsulated drug through processessuch as gel chromatography or ultrafiltration. The Liposomes are thentypically sterile filtered to remove any microorganisms which may bepresent in the suspension. Microorganisms may also be removed throughaseptic processing.

If one wishes to encapsulate large hydrophilic molecules with Liposomes,larger unilamellar vesicles can be formed by methods such as thereverse-phase evaporation (REV) or solvent infusion methods. Otherstandard methods for the formation of Liposomes are known in the art,for example, methods for the commercial production of Liposomes includethe homogenization procedure described in U.S. Pat. No. 4,753,788 andthe thin-film evaporation method described in U.S. Pat. No. 4,935,171,which are incorporated herein by reference.

It is to be understood that the therapeutic or diagnostic agent can alsobe associated with the surface of the Lipid Bilayer Vesicle. Forexample, a drug can be attached to a phospholipid or glyceride (aprodrug). The phospholipid or glyceride portion of the prodrug can beincorporated into the lipid bilayer of the Liposome by inclusion in thelipid formulation or loading into preformed Liposomes (see U.S. Pat.Nos. 5,194,654 and 5,223,263, which are incorporated by referenceherein).

It is readily apparent to one skilled in the art that the particularLiposome preparation method will depend on the intended use and the typeof lipids used to form the bilayer membrane.

Lee and Low (1994, JBC 269:3198-3204) and DeFrees et al. (1996, JACS118:6101-6104) first showed that co-formulation of ligand-PEG-lipid withlipid components gave liposomes with both inward and outward facingorientations of the PEG-ligand. Passive anchoring was outlined byZalipsky et al. (1997, Bioconj. Chem. 8:111-118) as a method foranchoring oligopeptide and oligosaccharide ligands exclusively to theexternal surface of liposomes. The central concept presented in theirwork is that oligo-PEG-lipid conjugates can be prepared and thenformulated into pre-formed liposomes via spontaneous incorporation(“anchoring”) of the lipid tail into the existing lipid bilayer. Thelipid group undergoes this insertion in order to reach a lower freeenergy state via the removal of its hydrophobic lipid anchor fromaqueous solution and its subsequent positioning in the hydrophobic lipidbilayer. The key advantage to such a system is that the oligo-lipid isanchored exclusively to the exterior of the lipid bilayer. Thus, nooligo-lipids are wasted by being unavailable for interactions with theirbiological targets by being in an inward-facing orientation.

The efficiency of delivery of a PDGF Nucleic Acid Ligand to cells may beoptimized by using lipid formulations and conditions known to enhancefusion of Liposomes with cellular membranes. For example, certainnegatively charged lipids such as phosphatidylglycerol andphosphatidylserine promote fusion, especially in the presence of otherfusogens (e.g., multivalent cations like Ca2+, free fatty acids, viralfusion proteins, short chain PEG, lysolecithin, detergents andsurfactants). Phosphatidylethanolamine may also be included in theLiposome formulation to increase membrane fusion and, concomitantly,enhance cellular delivery. In addition, free fatty acids and derivativesthereof, containing, for example, carboxylate moieties, may be used toprepare pH-sensitive Liposomes which are negatively charged at higher pHand neutral or protonated at lower pH. Such pH-sensitive Liposomes areknown to possess a greater tendency to fuse.

In the preferred embodiment, the PDGF Nucleic Acid Ligands of thepresent invention are derived from the SELEX methodology. SELEX isdescribed in U.S. patent application Ser. No. 07/536,428, entitledSystematic Evolution of Ligands by Exponential Enrichment, nowabandoned, U.S. patent application Ser. No. 07/714,131, filed Jun. 10,1991, entitled Nucleic Acid Ligands, now U.S. Pat. No. 5,475,096, andU.S. patent application Ser. No. 07/931,473, filed Aug. 17, 1992,entitled Methods for Identifying Nucleic Acid Ligands, now U.S. Pat. No.5,270,163 (see also WO 91/19813). These applications, each specificallyincorporated herein by reference, are collectively called the SELEXPatent Applications.

The SELEX process provides a class of products which are Nucleic Acidmolecules, each having a unique sequence, and each of which has theproperty of binding specifically to a desired Target compound ormolecule. Target molecules are preferably proteins, but can also includeamong others carbohydrates, peptidoglycans and a variety of smallmolecules. SELEX methodology can also be used to Target biologicalstructures, such as cell surfaces or viruses, through specificinteraction with a molecule that is an integral part of that biologicalstructure.

In its most basic form, the SELEX process may be defined by thefollowing series of steps:

1) A Candidate Mixture of Nucleic Acids of differing sequence isprepared. The Candidate Mixture generally includes regions of fixedsequences (i.e., each of the members of the Candidate Mixture containsthe same sequences in the same location) and regions of randomizedsequences. The fixed sequence regions are selected either: (a) to assistin the amplification steps described below, (b) to mimic a sequenceknown to bind to the Target, or (c) to enhance the concentration of agiven structural arrangement of the Nucleic Acids in the CandidateMixture. The randomized sequences can be totally randomized (i.e., theprobability of finding a base at any position being one in four) or onlypartially randomized (e.g., the probability of finding a base at anylocation can be selected at any level between 0 and 100 percent).

2) The Candidate Mixture is contacted with the selected Target underconditions favorable for binding between the Target and members of theCandidate Mixture. Under these circumstances, the interaction betweenthe Target and the Nucleic Acids of the Candidate Mixture can beconsidered as forming Nucleic Acid-target pairs between the Target andthose Nucleic Acids having the strongest affinity for the Target.

3) The Nucleic Acids with the highest affinity for the target arepartitioned from those Nucleic Acids with lesser affinity to the target.Because only an extremely small number of sequences (and possibly onlyone molecule of Nucleic Acid) corresponding to the highest affinityNucleic Acids exist in the Candidate Mixture, it is generally desirableto set the partitioning criteria so that a significant amount of theNucleic Acids in the Candidate Mixture (approximately 5-50%) areretained during partitioning.

4) Those Nucleic Acids selected during partitioning as having therelatively higher affinity for the target are then amplified to create anew Candidate Mixture that is enriched in Nucleic Acids having arelatively higher affinity for the target.

5) By repeating the partitioning and amplifying steps above, the newlyformed Candidate Mixture contains fewer and fewer unique sequences, andthe average degree of affinity of the Nucleic Acids to the target willgenerally increase. Taken to its extreme, the SELEX process will yield aCandidate Mixture containing one or a small number of unique NucleicAcids representing those Nucleic Acids from the original CandidateMixture having the highest affinity to the target molecule.

The basic SELEX method has been modified to achieve a number of specificobjectives. For example, U.S. patent application Ser. No. 07/960,093,filed Oct. 14, 1992, entitled “Method for Selecting Nucleic Acids on theBasis of Structure,” describes the use of SELEX in conjunction with gelelectrophoresis to select Nucleic Acid molecules with specificstructural characteristics, such as bent DNA. U.S. patent applicationSer. No. 08/123,935, filed Sep. 17, 1993, entitled “Photoselection ofNucleic Acid Ligands,” describes a SELEX based method for selectingNucleic Acid Ligands containing photoreactive groups capable of bindingand/or photocrosslinking to and/or photoinactivating a target molecule.U.S. patent application Ser. No. 08/134,028, filed Oct. 7, 1993,entitled “High-Affinity Nucleic Acid Ligands That Discriminate BetweenTheophylline and Caffeine,” now abandoned (see U.S. Pat. No. 5,580,737),describes a method for identifying highly specific Nucleic Acid Ligandsable to discriminate between closely related molecules, termedCounter-SELEX. U.S. patent application Ser. No. 08/143,564, filed Oct.25, 1993, entitled “Systematic Evolution of Ligands by ExponentialEnrichment: Solution SELEX,” now abandoned (see U.S. Pat. No.5,567,588), describes a SELEX-based method which achieves highlyefficient partitioning between oligonucleotides having high and lowaffinity for a target molecule. U.S. patent application Ser. No.07/964,624, filed Oct. 21, 1992, entitled “Nucleic Acid Ligands toHIV-RT and HIV-1 Rev,” now U.S. Pat. No. 5,496,938, describes methodsfor obtaining improved Nucleic Acid Ligands after SELEX has beenperformed. U.S. patent application Ser. No. 08/400,440, filed Mar. 8,1995, entitled “Systematic Evolution of Ligands by ExponentialEnrichment: Chemi-SELEX,” now U.S. Pat. No. 5,707,337, describes methodsfor covalently linking a ligand to its target.

The SELEX method encompasses the identification of high-affinity NucleicAcid Ligands containing modified nucleotides conferring improvedcharacteristics on the ligand, such as improved in vivo stability orimproved delivery characteristics. Examples of such modificationsinclude chemical substitutions at the ribose and/or phosphate and/orbase positions. SELEX-identified Nucleic Acid Ligands containingmodified nucleotides are described in U.S. patent application Ser. No.08/117,991, filed Sep. 8, 1993, entitled “High Affinity Nucleic AcidLigands Containing Modified Nucleotides,” now U.S. Pat. No. 5,660,985,that describes oligonucleotides containing nucleotide derivativeschemically modified at the 5- and 2′-positions of pyrimidines. U.S.patent application Ser. No. 08/134,028, supra, describes highly specificNucleic Acid Ligands containing one or more nucleotides modified with2′-amino (2′-NH₂), 2′-fluoro (2′-F), and/or 2′-O-methyl (2′-OMe). U.S.patent application Ser. No. 08/264,029, filed Jun. 22, 1994, entitled“Novel Method of Preparation of Known and Novel 2′ Modified Nucleosidesby Intramolecular Nucleophilic Displacement,” describes oligonucleotidescontaining various 2′-modified pyrimidines.

The SELEX method encompasses combining selected oligonucleotides withother selected oligonucleotides and non-oligonucleotide functional unitsas described in U.S. patent application Ser. No. 08/284,063, filed Aug.2, 1994, entitled “Systematic Evolution of Ligands by ExponentialEnrichment: Chimeric SELEX,” now U.S. Pat. No. 5,637,459, and U.S.patent application Ser. No. 08/234,997, filed Apr. 28, 1994, entitled“Systematic Evolution of Ligands by Exponential Enrichment: BlendedSELEX,” now U.S. Pat. No. 5,683,867, respectively. These applicationsallow the combination of the broad array of shapes and other properties,and the efficient amplification and replication properties, ofoligonucleotides with the desirable properties of other molecules.

The SELEX method further encompasses combining selected Nucleic AcidLigands with Lipophilic Compounds or Non-Immunogenic, High MolecularWeight Compounds in a diagnostic or therapeutic Complex as described inU.S. patent application Ser. No. 08/434,465, filed May 4, 1995, entitled“Nucleic Acid Ligand Complexes,” now U.S. Pat. No. 6,011,021. The SELEXmethod further encompasses combining selected VEGF Nucleic Acid Ligandswith lipophilic compounds, such as diacyl glycerol or dialkyl glycerol,as described in United States patent application Ser. No. 08/739,109,filed Oct. 25, 1996, entitled “Vascular Endothelial Growth Factor (VEGF)Nucleic Acid Ligand Complexes,” now U.S. Pat. No. 5,859,228. VEGFNucleic Acid Ligands that are associated with a High Molecular Weight,Non-Immunogenic Compound, such as Polyethyleneglycol, or a LipophilicCompound, such as Glycerolipid, phospholipid, or glycerol amide lipid,in a diagnostic or therapeutic complex are described in U.S. patentapplication Ser. No. 08/897,351, filed Jul. 21, 1997, entitled “VascularEndothelial Growth Factor (VEGF) Nucleic Acid Ligand Complexes,” nowU.S. Pat. No. 6,051,698. Each of the above described patent applicationswhich describe modifications of the basic SELEX procedure arespecifically incorporated by reference herein in their entirety.

SELEX identifies Nucleic Acid Ligands that are able to bind targets withhigh affinity and with outstanding specificity, which represents asingular achievement that is unprecedented in the field of Nucleic Acidsresearch. These characteristics are, of course, the desired propertiesone skilled in the art would seek in a therapeutic or diagnostic ligand.

In order to produce Nucleic Acid Ligands desirable for use as apharmaceutical, it is preferred that the Nucleic Acid Ligand (1) bindsto the target in a manner capable of achieving the desired effect on thetarget; (2) be as small as possible to obtain the desired effect; (3) beas stable as possible; and (4) be a specific ligand to the chosentarget. In most situations, it is preferred that the Nucleic Acid Ligandhas the highest possible affinity to the target. Additionally, NucleicAcid Ligands can have facilitating properties.

In commonly assigned U.S. patent application Ser. No. 07/964,624, filedOct. 21, 1992 ('624), now U.S. Pat. No.5,496,938, methods are describedfor obtaining improved Nucleic Acid Ligands after SELEX has beenperformed. The '624 application, entitled Nucleic Acid Ligands to HIV-RTand HIV- 1 Rev, is specifically incorporated herein by reference.

The SELEX process has been used to identify a group of high affinity RNALigands to PDGF from random ssDNA libraries and2′-fluoro-2′-deoxypyrimidine RNA ligands from random ssDNA libraries(U.S. patent application Ser. No. 08/618,693, filed Mar. 20, 1996,entitled High-Affinity PDGF Nucleic Acid Ligands, now U.S. Pat. No.5,723,594, which is a Continuation-in-Part Application of U.S. patentapplication Ser. No. 08/479,783, filed Jun. 7, 1995, entitledHigh-Affinity PDGF Nucleic Acid Ligands, now U.S. Pat. No. 5,668,264,and U.S. patent application Ser. No. 08/479,725, filed Jun. 7, 1995,entitled “High Affinity PDGF Nucleic Acid Ligands, now U.S. Pat. No.5,674,685, both of which are incorporated herein by reference; see alsoGreen et al. (1995) Chemistry and Biology 2:683-695).

In embodiments where the PDGF Nucleic Acid Ligand(s) can serve in atargeting capacity, the PDGF Nucleic Acid Ligands adopt a threedimensional structure that must be retained in order for the PDGFNucleic Acid Ligand to be able to bind its target. In embodiments wherethe Lipid Construct comprises a Complex and the PDGF Nucleic Acid Ligandof the Complex is projecting from the surface of the Lipid Construct,the PDGF Nucleic Acid Ligand must be properly oriented with respect tothe surface of the Lipid Construct so that its target binding capacityis not compromised. This can be accomplished by attaching the PDGFNucleic Acid Ligand at a position that is distant from the bindingportion of the PDGF Nucleic Acid Ligand. The three dimensional structureand proper orientation can also be preserved by use of a Linker orSpacer as described supra.

Any variety of therapeutic or diagnostic agents can be attached to theComplex for targeted delivery by the Complex. In addition, any varietyof therapeutic or diagnostic agents can be attached encapsulated, orincorporated into the Lipid Construct as discussed supra for targeteddelivery by the Lipid Construct.

In embodiments where the Complex is comprised of a Lipophilic Compoundand a PDGF Nucleic Acid Ligand in association with a Liposome, forexample, the PDGF Nucleic Acid Ligand could target tumor cellsexpressing PDGF(e.g., in Kaposi's sarcoma) for delivery of an antitumordrug (e.g., daunorubicin) or imaging agent (e.g., radiolabels). Itshould be noted that cells and tissues surrounding the tumor may alsoexpress PDGF, and targeted delivery of an antitumor drug to these cellswould also be effective.

In an alternative embodiment, the therapeutic or diagnostic agent to bedelivered to the Target cell could be another Nucleic Acid Ligand.

It is further contemplated by this invention that the agent to bedelivered can be incorporated into the Complex in such a way as to beassociated with the outside surface of the Liposome (e.g., a prodrug,receptor antagonist, or radioactive substance for treatment or imaging).As with the PDGF Nucleic Acid Ligand, the agent can be associatedthrough covalent or Non-Covalent Interactions. The Liposome wouldprovide targeted delivery of the agent extracellularly, with theLiposome serving as a Linker.

In another embodiment, a Non-Immunogenic, High Molecular Weight Compound(e.g., PEG) can be attached to the Liposome to provide ImprovedPharmacokinetic Properties for the Complex. PDGF Nucleic Acid Ligandsmay be attached to the Liposome membrane or may be attached to aNon-Immunogenic, High Molecular Weight Compound which in turn isattached to the membrane. In this way, the Complex may be shielded fromblood proteins and thus be made to circulate for extended periods oftime while the PDGF Nucleic Acid Ligand is still sufficiently exposed tomake contact with and bind to its Target.

In another embodiment of the present invention, more than one PDGFNucleic Acid Ligand is attached to the surface of the same Liposome.This provides the possibility of bringing the same PDGF molecules inclose proximity to each other and can be used to generate specificinteractions between the PDGF molecules.

In an alternative embodiment of the present invention, PDGF Nucleic AcidLigands and a Nucleic Acid Ligand to a different Target can be attachedto the surface of the same Liposome. This provides the possibility ofbringing PDGF in close proximity to a different Target and can be usedto generate specific interactions between PDGF and the other Target. Inaddition to using the Liposome as a way of bringing Targets in closeproximity, agents could be encapsulated in the Liposome to increase theintensity of the interaction.

The Lipid Construct comprising a Complex allows for the possibility ofmultiple binding interactions to PDGF. This, of course, depends on thenumber of PDGF Nucleic Acid Ligands per Complex, and the number ofComplexes per Lipid Construct, and mobility of the PDGF Nucleic AcidLigands and receptors in their respective membranes. Since the effectivebinding constant may increase as the product of the binding constant foreach site, there is a substantial advantage to having multiple bindinginteractions. In other words, by having many PDGF Nucleic Acid Ligandsattached to the Lipid Construct, and therefore creating multivalency,the effective affinity (i.e., the avidity) of the multimeric Complex forits Target may become as good as the product of the binding constant foreach site.

In certain embodiments of the invention, the Complex of the presentinvention is comprised of a PDGF Nucleic Acid Ligand attached to aLipophilic Compound. In this case, the pharmacokinetic properties of theComplex will be improved relative to the PDGF Nucleic Acid Ligand alone.As discussed supra, the Lipophilic Compound may be covalently bound tothe PDGF Nucleic Acid Ligand at numerous positions on the PDGF NucleicAcid Ligand.

In another embodiment of the invention, the Lipid Construct comprises aPDGF Nucleic Acid Ligand or Complex. In this embodiment, theglycerolipid can assist in the incorporation of the PDGF Nucleic AcidLigand into the Liposome due to the propensity for a glycerolipid toassociate with other Lipophilic Compounds. The glycerolipid inassociation with a PDGF Nucleic Acid Ligand can be incorporated into thelipid bilayer of the Liposome by inclusion in the formulation or byloading into preformed Liposomes. The glycerolipid can associate withthe membrane of the Liposome in such a way so as the PDGF Nucleic AcidLigand is projecting into or out of the Liposome. In embodiments wherethe PDGF Nucleic Acid Ligand is projecting out of the Complex, the PDGFNucleic Acid Ligand can serve in a targeting capacity. It is to beunderstood that additional compounds can be associated with the LipidConstruct to further improve the Pharmacokinetic Properties of the LipidConstruct. For example, a PEG may be attached to the exterior-facingpart of the membrane of the Lipid Construct.

In other embodiments, the Complex of the present invention is comprisedof a PDGF Nucleic Acid Ligand covalently linked to a Non-Immunogenic,High Molecular Weight Compound such as Polyalkylene Glycol or PEG. Inthis embodiment, the pharmacokinetic properties of the Complex areimproved relative to the PDGF Nucleic Acid Ligand alone. ThePolyalkylene Glycol or PEG may be covalently bound to a variety ofpositions on the PDGF Nucleic Acid Ligand. In embodiments wherePolyalkylene Glycol or PEG are used, it is preferred that the PDGFNucleic Acid Ligand is bonded through the 5′ hydroxyl group via aphosphodiester linkage.

In certain embodiments, a plurality of Nucleic Acid Ligands can beassociated with a single Non-Immunogenic, High Molecular WeightCompound, such as Polyalkylene Glycol or PEG, or a Lipophilic Compound,such as a glycerolipid. The Nucleic Acid Ligands can all be to PDGF orPDGF and a different Target. In embodiments where there are multiplePDGF Nucleic Acid Ligands, there is an increase in avidity due tomultiple binding interactions with PDGF. In yet further embodiments, aplurality of Polyalkylene Glycol, PEG, glycerol lipid molecules can beattached to each other. In these embodiments, one or more PDGF NucleicAcid Ligands or Nucleic Acid Ligands to PDGF and other Targets can beassociated with each Polyalkylene Glycol, PEG, or glycerol lipid. Thisalso results in an increase in avidity of each Nucleic Acid Ligand toits Target. In embodiments where multiple PDGF Nucleic Acid Ligands areattached to Polyalkylene Glycol, PEG, or glycerol lipid, there is thepossibility of bringing PDGF molecules in close proximity to each otherin order to generate specific interactions between PDGF. Where multipleNucleic Acid Ligands specific for PDGF and different Targets areattached to Polyalkylene Glycol, PEG, or glycerol lipid, there is thepossibility of bringing PDGF and another Target in close proximity toeach other in order to generate specific interactions between the PDGFand the other Target. In addition, in embodiments where there areNucleic Acid Ligands to PDGF or Nucleic Acid Ligands to PDGF anddifferent Targets associated with Polyalkylene Glycol, PEG, or glycerollipid, a drug can also be associated with Polyalkylene Glycol, PEG, orglycerol lipid. Thus the Complex would provide targeted delivery of thedrug, with Polyalkylene Glycol, PEG, or glycerol lipid serving as aLinker.

PDGF Nucleic Acid Ligands selectively bind PDGF. Thus, a Complexcomprising a PDGF Nucleic Acid Ligand and a Non-Immunogenic, HighMolecular Weight Compound or Lipophilic Compound or a Lipid Constructcomprising a PDGF Nucleic Acid Ligand or a Complex are useful aspharmaceuticals or diagnostic agents. The PDGF Nucleic AcidLigand-containing Complexes and Lipid Constructs can be used to treat,inhibit, prevent or diagnose any disease state that involvesinappropriate PDGF production, for example, cancer, angiogenesis,restenosis, and fibrosis. PDGF is produced and secreted in varyingamounts by many tumor cells. Thus, the present invention, includesmethods of treating, inhibiting, preventing, or diagnosing cancer byadministration of a Complex comprising a PDGF Nucleic Acid Ligand and aNon-Immunogenic, High Molecular Weight Compound or Lipophilic Compound,a Lipid Construct comprising a Complex, or a PDGF Nucleic Acid Ligand inassociation with a Lipid Construct without being part of the Complex.

Angiogenesis rarely occurs in healthy adults, except during themenstrual cycle and wound healing. Angiogenesis is a central feature,however, of various disease states, including, but not limited tocancer, diabetic retinopathy, macular degeneration, psoriasis andrheumatoid arthritis. The present invention, therefore, includes methodsof treating, inhibiting, preventing, or diagnosing angiogenesis byadministration of a Complex comprising PDGF Nucleic Acid Ligand and aNon-Immunogenic, High Molecular Weight Compound or Lipophilic Compound,a Lipid Construct comprising PDGF Nucleic Acid Ligand or a Complexcomprising a PDGF Nucleic Acid Ligand and a Non-Immunogenic, HighMolecular Weight Compound or Lipophilic Compound.

PDGF is also produced in fibrosis in organs, such as lung, bone marrowand kidney. Fibrosis can also be associated with radiation treatments.The present invention, therefore, includes methods of treating,inhibiting, preventing or diagnosing lung, bone marrow, kidney andradiation treatment-associated fibrosis by administration of a Complexcomprising PDGF Nucleic Acid Ligand and a Non-Immunogenic, HighMolecular Weight Compound or Lipophilic Compound, a Lipid Constructcomprising PDGF Nucleic Acid Ligand or a Complex comprising a PDGFNucleic Acid Ligand and a Non-Immunogenic, High Molecular WeightCompound or Lipophilic Compound.

PDGF is a prominent growth factor involved in restenosis. Restenosis,the reocclusion of a diseased blood vessel after treatment to eliminatestenosis, is a common occurrence that develops following coronaryinterventions and some peripheral vessel interventions. Additionally,stents have been used in the treatment of or in conjunction withtreatment of coronary and non-coronary vessels; however, restenosis isalso associated with use of stents (called in-stent restenosis).In-stent restenosis occurs in about 15-30% of coronary interventions andfrequently in some peripheral vessel interventions. For example,in-stent restenosis is a significant problem in small vessels, withfrequencies ranging from 15% to 40% in stented femoral or poplitealarteries. Intermediate-sized vessels, such as renal arteries, have anin-stent restenosis rate of 10-20%.

The present invention, therefore, includes methods of treating,inhibiting, preventing or diagnosing restenosis by administration of aComplex comprising PDGF Nucleic Acid Ligand and a Non-Immunogenic, HighMolecular Weight Compound or Lipophilic Compound, a Lipid Constructcomprising PDGF Nucleic Acid Ligand or a Complex comprising a PDGFNucleic Acid Ligand and a Non-Immunogenic, High Molecular WeightCompound or Lipophilic Compound. The present invention also includesmethods of treating, inhibiting, preventing or diagnosing restenosis incoronary and non-coronary vessels. The present invention also includesmethods of treating, inhibiting, preventing or diagnosing in-stentrestenosis.

Additionally, cancer, angiogenesis, restenosis, and fibrosis involve theproduction of growth factors other than PDGF. Thus, it is contemplatedby this invention that a Complex comprising PDGF Nucleic Acid Ligand anda Non-Immunogenic, High Molecular Weight Compound or LipophilicCompound, a Lipid Construct comprising PDGF Nucleic Acid Ligand or aComplex comprising a PDGF Nucleic Acid Ligand and a Non-Immunogenic,High Molecular Weight Compound or Lipophilic Compound can be used inconjunction with Complexes comprising Nucleic Acid Ligands to othergrowth factors (such as bFGF, TGFβ, hKGF, etc.) and a Non-Immunogenic,High Molecular Weight Compound or Lipophilic Compound, a Lipid Constructcomprising PDGF Nucleic Acid Ligand or a Complex comprising a PDGFNucleic Acid Ligand and a Non-Immunogenic, High Molecular WeightCompound or Lipophilic Compound.

In one embodiment of the present invention, the Lipid Constructcomprises a Complex comprised of a PDGF Nucleic Acid Ligand and aLipophilic Compound with an additional diagnostic or therapeutic agentencapsulated in the Lipid Construct or associated with the interior ofthe Lipid Construct. In the preferred embodiment, the Lipid Construct isa Lipid Bilayer Vesicle, and more preferably a Liposome. The therapeuticuse of Liposomes includes the delivery of drugs which are normally toxicin the free form. In the liposomal form, the toxic drug is occluded, andmay be directed away from the tissues sensitive to the drug and targetedto selected areas. Liposomes can also be used therapeutically to releasedrugs over a prolonged period of time, reducing the frequency ofadministration. In addition, liposomes can provide a method for formingaqueous dispersions of hydrophobic or amphiphilic drugs, which arenormally unsuitable for intravenous delivery.

In order for many drugs and imaging agents to have therapeutic ordiagnostic potential, it is necessary for them to be delivered to theproper location in the body, and the liposome can thus be readilyinjected and form the basis for sustained release and drug delivery tospecific cell types, or parts of the body. Several techniques can beemployed to use liposomes to target encapsulated drugs to selected hosttissues, and away from sensitive tissues. These techniques includemanipulating the size of the liposomes, their net surface charge, andtheir route of administration. MLVs, primarily because they arerelatively large, are usually rapidly taken up by thereticuloendothelial system (principally the liver and spleen). UVs, onthe other hand, have been found to exhibit increased circulation times,decreased clearance rates and greater biodistribution relative to MLVs.

Passive delivery of liposomes involves the use of various routes ofadministration, e.g., intravenous, subcutaneous, intramuscular andtopical. Each route produces differences in localization of theliposomes. Two common methods used to direct liposomes actively toselected target areas involve attachment of either antibodies orspecific receptor ligands to the surface of the liposomes. In oneembodiment of the present invention, the PDGF Nucleic Acid Ligand isassociated with the outside surface of the liposome, and serves in atargeting capacity. Additional targeting components, such as antibodiesor specific receptor ligands can be included on the liposome surface, aswould be known to one of skill in the art. In addition, some effortshave been successful in targeting liposomes to tumors without the use ofantibodies, see, for example, U.S. Pat. No. 5,019,369, U.S. Pat. No.5,435,989, and U.S. Pat. No. 4,441,775, and it would be known to one ofskill in the art to incorporate these alternative targeting methods.

Therapeutic or diagnostic compositions of a Complex comprising a PDGFNucleic Acid Ligand and a Non-Immunogenic, High Molecular WeightCompound or Lipophilic Compound, a Lipid Construct comprising a Complexcomprised of a PDGF Nucleic Acid Ligand and a Non-Immunogenic, HighMolecular Weight Compound or Lipophilic Compound, and a PDGF NucleicAcid Ligand in association with a Lipid Construct without being part ofa Complex may be administered parenterally by injection, although othereffective administration forms, such as intraarticular injection,inhalant mists, orally active formulations, transdermal iotophoresis orsuppositories, are also envisioned. They may also be applied locally bydirect injection, can be released from devices, such as implanted stentsor catheters, or delivered directly to the site by an infusion pump. Onepreferred carrier is physiological saline solution, but it iscontemplated that other pharmaceutically acceptable carriers may also beused. In one embodiment, it is envisioned that the carrier and the PDGFNucleic Acid Ligand Complex constitute a physiologically-compatible,slow release formulation. The primary solvent in such a carrier may beeither aqueous or non-aqueous in nature. In addition, the carrier maycontain other pharmacologically-acceptable excipients for modifying ormaintaining the pH, osmolarity, viscosity, clarity, color, sterility,stability, rate of dissolution, or odor of the formulation. Similarly,the carrier may contain still other pharmacologically-acceptableexcipients for modifying or maintaining the stability, rate ofdissolution, release, or absorption of the PDGF Nucleic Acid Ligand.Such excipients are those substances usually and customarily employed toformulate dosages for parental administration in either unit dose ormulti-dose form.

Once the therapeutic or diagnostic composition has been formulated, itmay be stored in sterile vials as a solution, suspension, gel, emulsion,solid, or dehydrated or lyophilized powder. Such formulations may bestored either in ready to use form or requiring reconstitutionimmediately prior to administration. The manner of administeringformulations containing PDGF Nucleic Acid Ligand for systemic deliverymay be via subcutaneous, intramuscular, intravenous, intranasal orvaginal or rectal suppository.

The advantages of the Complexes and Lipid Constructs of the inventioninclude: i) improving the plasma pharmacokinetics of the Nucleic AcidLigand; ii) presenting Nucleic Acid Ligands in a multivalent array withthe aim of increasing the avidity of interaction with their targets;iii) combining two or more presenting Nucleic Acid Ligands withdifferent specificities in the same liposome particle; iv) enhancing thedelivery of presenting Nucleic Acid Ligands to tumors by takingadvantage of the intrinsic tumor targeting properties of liposomes; andv) using the high affinity and specificity of presenting Nucleic AcidLigands, which is comparable to that of antibodies, to guide liposomalcontents to specific targets. Presenting Nucleic Acid Ligands are wellsuited for the kinds of preparations described here since, unlike mostproteins, the denaturation of presenting Nucleic Acid Ligands by heat,various molecular denaturants and organic solvents is readilyreversible.

The following examples are provided to explain and illustrate thepresent invention and are not to be taken as limiting of the invention.Example 1 describes the various materials and experimental proceduresused in Examples 2-4 for the generation of ssDNA ligands to PDGF andtests associated therewith. Example 2 describes the ssDNA ligands toPDGF and the predicted secondary structure of selected nucleic acidligands and a shared secondary structure motif. Example 3 describes theminimum sequence necessary for high affinity binding, the sites on thenucleic acid ligands and PDGF that are in contact, inhibition by DNAligands of PDGF isoforms on cultured cells, and inhibition of mitogeniceffects of PDGF in cells by DNA ligands. Example 4 describessubstitutions of SELEX-derived ligands with modified nucleotides.Example 5 describes synthesis of PEG-modified PDGF Nucleic Acid Ligands.Example 6 describes stability of modified ligands in serum. Example 7describes efficacy of a modified ligand (NX31975-40K PEG) in restenosis.Example 8 describes the various materials and method used in Example 9for testing the inhibition of PDGF in glomerulonephritis. Example 9describes inhibition of PDGF in glomerulonephritis. Example 10 describesthe experimental procedures for evolving 2′-fluoro-2′-deoxypyrimidineRNA ligands to PDGF and the RNA sequences obtained.

EXAMPLE 1 EXPERIMENTAL PROCEDURES

This example provides the general procedures followed and incorporatedin

EXAMPLES 2-4

MATERIALS.

Recombinant human PDGF-AA (Mr=29,000), PDGF-AB (Mr=27,000) and PDGF-BB(Mr=25,000) were purchased from R&D Systems (Minneapolis, Minn.) inlyophilized form, free from carrier protein. All three isoforms wereproduced in E. coli from synthetic genes based on the sequences for thelong form of the mature human PDGF A-chain (Betsholtz et al. (1986)Nature 320:695-699) and the naturally occurring mature form of humanPDGF B-chain (Johnsson et al. (1984) EMBO J. 3:921-928). Randomized DNAlibraries, PCR primers and DNA ligands and5′-iodo-2′-deoxyuridine-substituted DNA ligands were synthesized byNeXstar Pharmaceuticals, Inc. (Boulder, Colo.) or by Operon Technologies(Alameda, Calif.) using the standard solid phase phosphoramidite method(Sinha et al. (1984) Nucleic Acids Res. 12:4539-4557).

SINGLE STRANDED DNA (ssDNA) SELEX

Essential features of the SELEX procedure have been described in detailin the SELEX patent Applications (see also Tuerk and Gold (1990) Science249:505; Jellinek et al. (1994) Biochemistry 33:10450; Jellinek et al.(1993) Proc. Natl. Acad. Sci. USA 90:1227), which are incorporated byreference herein. The initial ssDNA library containing a contiguousrandomized region of forty nucleotides, flanked by primer annealingregions (Table 1) (SEQ ID NOS: 1-3) of invariant sequence, wassynthesized by the solid phase phosphoramidite method using equal molarmixture of the four phosphoramidites to generate the randomizedpositions. The ssDNA library was purified by electrophoresis on an 8%polyacrylamide/7 M urea gel. The band that corresponds to thefull-length DNA was visualized under UV light, excised from the gel,eluted by the crush and soak method, ethanol precipitated and pelletedby centrifugation. The pellet was dried under vacuum and resuspended inphosphate buffered saline supplemented with 1 mM MgCl₂ (PBSM=10.1 mMNa₂HPO₄, 1.8 mM KH₂PO₄, 137 mM NaCl and 2.7 mM KCl, 1 mM MgCl₂, pH 7.4)buffer. Prior to incubation with the protein, the ssDNA was heated at90° C. for 2 minutes in PBSM and cooled on ice. The first selection wasinitiated by incubating approximately 500 pmol (3×10¹⁴ molecules) of 5′³²P end-labeled random ssDNA with PDGF-AB in binding buffer (PBSMcontaining 0.01% human serum albumin (HSA)). The mixture was incubatedat 4° C. overnight, followed by a brief (15 min) incubation at 37° C.The DNA bound to PDGF-AB was separated from unbound DNA byelectrophoresis on an 8% polyacrylamide gel (1:30bis-acrylamide:acrylamide) at 4° C. and at 5 V/cm with 89 mM Tris-borate(pH 8.3) containing 2 mM EDTA as the running buffer. The band thatcorresponds to the PDGF-ssDNA complex, which runs with about half theelectrophoretic mobility of the free ssDNA, was visualized byautoradiography, excised from the gel and eluted by the crush and soakmethod. In subsequent affinity selections, the ssDNA was incubated withPDGF-AB for 15 minutes at 37° C. in binding buffer and the PDGF-boundssDNA was separated from the unbound DNA by nitrocellulose filtration,as previously described (Green et al. (1995) Chemistry and Biology2:683-695). All affinity-selected ssDNA pools were amplified by PCR inwhich the DNA was subjected to 12-20 rounds of thermal cycling (30 s at93° C., 10 s at 52° C., 60 s at 72° C.) in 10 mM Tris-Cl (pH 8.4)containing 50 mM KCl, 7.5 mM MgCl₂, 0.05 mg/ml bovine serum albumin, 1mM deoxynucleoside triphosphates, 5 μM primers (Table 1) (SEQ ID NOS: 2,3) and 0.1 units/μl Taq polymerase. The 5′ PCR primer was 5′ end-labeledwith polynucleotide kinase and [α-³²P]ATP and the 3′ PCR primer wasbiotinylated at the 5′ end using biotin phosphoramidite (Glen Research,Sterling, Va.). Following PCR amplification, streptavidin (Pierce,Rockford, Ill.) was added to the unpurified PCR reaction mixture at a10-fold molar excess over the biotinylated primer and incubated for 15min at room temperature. The dsDNA was denatured by adding an equalvolume of stop solution (90% formamide, 1% sodium dodecyl sulfate,0.025% bromophenol blue and xylene cyanol) and incubating for 20 min atroom temperature. The radiolabeled strand was separated from thestreptavidin-bound biotinylated strand by electrophoresis on 12%polyacrylamide/7M urea gels. The faster migrating radiolabeled(non-biotinylated) ssDNA strand was cut out of the gel and recovered asdescribed above. The amount of ssDNA was estimated from the absorbanceat 260 nm using the extinction coefficient of 33 μg/ml/absorbance unit(Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, 2nd Ed.3 vols., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).

Cloning and Sequencing. The amplified affinity-enriched pool from SELEXround 12 was purified on a 12% polyacrylamide gel and cloned betweenHindIII and PstI sites in JM109 strain of E. coli (Sambrook et al.(1989) Molecular Cloning: A Laboratory Manual, 2nd Ed. 3 vols., ColdSpring Harbor Laboratory Press, Cold Spring Harbor). Individual cloneswere used to prepare plasmids by alkaline lysis. Plasmids were sequencedat the insert region using the forward sequencing primer and Sequenase2.0 (Amersham, Arlington Heights, Ill.) according to the manufacturer'sprotocol.

Determination of the Apparent Equilibrium Dissociation Constants and theDissociation Rate Constants. The binding of ssDNA ligands at lowconcentrations to varying concentrations of PDGF was determined by thenitrocellulose filter binding method as described (Green et al. (1995)Chemistry and Biology 2:683-695). The concentrations of PDGF stocksolutions (in PBS) were determined from the absorbance readings at 280nm using the following _(e280) values calculated from the amino acidsequences (Gill and von Hippel (1989) Anal. Biochem. 182:319-326):19,500 M⁻¹ cm⁻¹ for PDGF-AA, 15,700 M⁻¹ cm⁻¹ for PDGF-AB and 11,800 M⁻¹cm⁻¹ for PDGF-BB. ssDNA for all binding experiments were purified byelectrophoresis on 8% (>80 nucleotides) or 12% (<40 nucleotides)polyacrylamide/7 M urea gels. All ssDNA ligands were heated at 90° C. inbinding buffer at high dilution (≈1 nM) for 2 min and cooled on iceprior to further dilution into the protein solution. The bindingmixtures were typically incubated for 15 min at 37° C. beforepartitioning on nitrocellulose filters.

The binding of DNA ligands (L) to PDGF-AA (P) is adequately describedwith the bimolecular binding model for which the fraction of bound DNAat equilibrium (q) is given by eq. 1,

q=(f/2[L]_(t)){[P]_(t)+[L]_(t)+K_(d)−[([P]_(t)+[L]_(t)+K_(d))²−4[P]_(t)[L]_(t)]^(½})  (1)

where [P]_(t) and [R]_(t) are total protein and total DNAconcentrations, K_(d) is the equilibrium dissociation constant and f isthe efficiency of retention of protein-DNA complexes on nitrocellulosefilters (Irvine et al. (1991) J. Mol. Biol. 222:739-761; Jellinek et al.(1993) Proc. Natl. Acad. Sci. USA 90:11227-11231).

The binding of DNA ligands to PDGF-AB and PDGF-BB is biphasic and can bedescribed by a model in which the DNA ligand is composed of twonon-interconverting components (L₁ and L₂) that bind to the protein withdifferent affinities, described by corresponding dissociation constants,K_(d1) and K_(d2) (Jellinek et al. (1993) Proc. Natl. Acad. Sci. USA90:11227-11231). In this case, the explicit solution for the fraction ofbound DNA (q) is given by eq. 2, $\begin{matrix}{{q = {{f\left( {\frac{\chi_{1}K_{d1}}{1 + {K_{d1}\lbrack P\rbrack}} + \frac{\chi_{2}K_{d2}}{1 + {K_{d2}\lbrack P\rbrack}}} \right)}\lbrack P\rbrack}}{{{with}\lbrack P\rbrack} = \frac{\lbrack P\rbrack t}{1 + \frac{\chi_{1}{K_{d1}\lbrack L\rbrack}_{t}}{1 + {K_{d1}\lbrack P\rbrack}} + \frac{\chi_{2}{K_{d2}\lbrack L\rbrack}_{t}}{1 + {K_{d2}\lbrack P\rbrack}}}}} & (2)\end{matrix}$

where X₁ and X₂(=1−X₁) are the mole fractions of L₁ and L₂. The K_(d)values for the binding of DNA ligands to PDGF were calculated by fittingthe data points to eq. 1 (for PDGF-AA) or eq. 2 (for PDGF-AB andPDGF-BB) using the non-linear least squares method.

The dissociation rate constants (k_(off)) were determined by measuringthe amount of ³²P 5′-end labeled minimal ligands (0.17 nM) bound toPDGF-AB (1 nM) as a function of time following the addition of 500-foldexcess of unlabeled ligands, using nitrocellulose filter binding as thepartitioning method. The k_(off) values were determined by fitting thedata points to the first-order rate equation (eq. 3)

(q−q_(∞))/(q_(o)−q_(∞))=exp(−k_(off)t)  (3)

where q, q_(o) and q_(∞) represent the fractions of DNA bound to PDGF-ABat any time (t), t=0 and t=∞, respectively.

Minimal Ligand Determinations. To generate a population of 5′end-labeled DNA ligands serially truncated from the 3′ end, a primercomplementary to the 3′ invariant sequence region of a DNA ligandtemplate (truncated primer 5N2, Table 1) (SEQ ID NO: 3) was radiolabeledat the 5′ end with [γ-³²P]-ATP and T4 polynucleotide kinase, annealed tothe template and extended with Sequenase (Amersham, Arlington Heights,Ill.) and a mixture of all four dNTPs and ddNTPs. Following incubationin binding buffer for 15 min at 37° C., the fragments from thispopulation that retain high affinity binding to PDGF-AB were separatedfrom those with weaker affinity by nitrocellulose filter partitioning.Electrophoretic resolution of the fragments on 8% polyacrylamide/7 Murea gels, before and after affinity selection, allows determination ofthe 3′ boundary. To generate a population of 3′ end-labeled DNA ligandsserially truncated from the 5′ end, the DNA ligands were radiolabeled atthe 3′ end with [α-³²P]-cordycepin-5′-triphosphate (New England Nuclear,Boston, Mass.) and T4 RNA ligase (Promega, Madison, Wis.),phosphorylated at the 5′ end with ATP and T4 polynucleotide kinase, andpartially digested with lambda exonuclease (Gibco BRL, Gaithersburg,Md.). Partial digestion of 10 pmols of 3′-labeled ligand was done in 100μL volume with 7 mM glycine-KOH (pH 9.4), 2.5mM MgCl₂, 1 μg/ml BSA, 15μg tRNA, and 4 units of lambda exonuclease for 15 min at 37°. The 5′boundary was determined in an analogous manner to that described for the3′ boundary.

Melting Temperature (T_(m)) Measurements. Melting profiles for theminimal DNA ligands were obtained on a Cary Model 1E spectrophotometer.Oligonucleotides (320-400 nM) were heated to 95° C. in PBS, PBSM or PBSwith 1 mM EDTA and cooled to room temperature prior to the meltingprofile determination. Melting profiles were generated by heating thesamples at the rate of 1° C./min from 15-95° C. and recording theabsorbance every 0.1° C. The first derivative of the data points wascalculated using the plotting program KaleidaGraph (Synergy Software,Reading, Pa.). The first derivative values were smoothed using a 55point smoothing function by averaging each point with 27 data points oneach side. The peak of the smoothed first derivative curves was used toestimate the T_(m) values.

Crosslinking of 5-iodo-2′-deoxyuridine-substituted DNA Ligands toPDGF-AB. DNA ligands containing single or multiple substitutions of5′-iodo-2′-deoxyuridine for thymidine were synthesized using the solidphase phosphoramidite method. To test for the ability to crosslink,trace amounts of 5′ ³²P end-labeled ligands were incubated with PDGF-AB(100 nM) in binding buffer at 37° C. for 15 min prior to irradiation.The binding mixture was transferred to a 1 cm path length cuvettethermostated at 37° C. and irradiated at 308 nm for 25-400 s at 20 Hzusing a XeCl charged Lumonics Model EX748 excimer laser. The cuvette waspositioned 24 cm beyond the focal point of a convergent lens, with theenergy at the focal point measuring 175 mjoules/pulse. Followingirradiation, aliquots were mixed with an equal volume of formamideloading buffer containing 0.1% SDS and incubated at 95° C. for 5 minprior to resolution of the crosslinked PDGF/ligand complex from the freeligand on 8% polyacrylamide/7 M urea gels.

To identify the protein site of crosslinking for ligand 20t-I4 (SEQ IDNO: 92), binding and irradiation were done on a larger scale. PDGF-ABand 5′ ³²P end-labeled ligand, each at 1 μM in PBSM, were incubated andirradiated (300 s) as described above in two 1 ml reaction vessels. Thereaction mixtures were combined, ethanol precipitated and resuspended in0.3 ml of Tris-HCl buffer (100 mM, pH 8.5). The PDGF-AB/ligandcrosslinked complex was digested with 0.17 μg/μl of modified trypsin(Boehringer Mannheim) for 20 hours at 37° C. The digest mixture wasextracted with phenol/chloroforn, chloroform and then ethanolprecipitated. The pellet was resuspended in water and an equal volume offormamide loading buffer with 5% (v/v) β-mercaptoethanol (no SDS),incubated at 95° C. for 5 min, and resolved on a 40 cm 8%polyacrylamide/7 M urea gel. The crosslinked tryptic-peptide/ligand thatmigrated as two closely spaced bands about 1.5 cm above the free ligandband was excised from the gel and eluted by the crush and soak methodand ethanol precipitated. The dried crosslinked peptide (about 160pmoles based on the specific activity) was sequenced by Edmandegradation (Midwest Analytical, Inc., St. Louis, Mo).

Receptor Binding Assay. The binding of ¹²⁵I-PDGF-AA and ¹²⁵I-PDGF-BB toporcine aortic endothelial (PAE) cells transfected with PDGF α-orβ-receptors were performed as described (Heldin et al., (1988) EMBO J.7:1387-1394). Different concentrations of DNA ligands were added to thecell culture (1.5 cm²) in 0.2 ml of phosphate buffered salinesupplemented with 1 mg bovine serum albumin per ml together with¹²⁵I-PDGF-AA (2 ng, 100,000 cpm) or ¹²⁵I-PDGF-BB (2 ng, 100,000 cpm).After incubation at 4° C. for 90 min, the cell cultures were washed andcell associated radioactivity determined in a g-counter (Heldin et al.,(1988) EMBO J. 7:1387-1394).

[³H]thymidine Incorporation Assay. The incorporation of [³H]thymidineinto PAE cells expressing PDGF β-receptor in response to 20 ng/ml ofPDGF-BB or 10% fetal calf serum and in the presence of differentconcentrations of DNA ligands was performed as described (Mori et al.(1991) J. Biol. Chem. 266:21158-21164). After incubation for 24 h at 37°C., ³H-radioactivity incorporated into DNA was determined using aβ-couonter.

EXAMPLE 2 ssDNA LIGANDS OF PDGF

High affinity DNA ligands to PDGF AB were identified by the SELEXprocess from a library of ≈3×10¹⁴ molecules (500 pmol) of singlestranded DNA randomized at forty contiguous positions (Table 1) (SEQ IDNO: 1). The PDGF-bound DNA was separated from unbound DNA bypolyacrylamide gel electrophoresis in the first round and bynitrocellulose filter binding in the subsequent rounds. After 12 roundsof SELEX, the affinity-enriched pool bound to PDGF-AB with an apparentdissociation constant (K_(d)) of ≈50 pM (data not shown). Thisrepresented an improvement in affinity of ≈700-fold compared to theinitial randomized DNA library. This affinity-enriched pool was used togenerate a cloning library from which 39 isolates were sequenced.Thirty-two of these ligands were found to have unique sequences (Table2) (SEQ ID NOS: 4-35). Ligands that were subjected to the minimalsequence determination are marked with an asterisk (*) next to the clonenumber. The clone numbers that were found to retain high affinitybinding as minimal ligands are italicized. All ligands shown in Table 2were screened for their ability to bind to PDGF AB using thenitrocellulose filter binding method. To identify the best ligands fromthis group, the relative affinities for PDGF-AB were determined bymeasuring the fraction of 5′ ³²P end-labeled ligands bound to PDGF-ABover a range of protein concentrations. For the ligands that bound toPDGF-AB with high affinity, the affinity toward PDGF-BB and PDGF-AA wasalso examined: in all cases, the affinity of ligands for PDGF-AB andPDGF-BB was comparable while the affinity for PDGF-AA was considerablylower (data not shown).

Twenty-one of the thirty-two unique ligands can be grouped into asequence family shown in Table 3 (SEQ ID NOS: 4, 5, 7-9, 14-24, 26, 31,32, 34 and 35). The sequences of the initially randomized region(uppercase letters) are aligned according to the consensus three-wayhelix junction motif. Nucleotides in the sequence-invariant region(lowercase letters) are only shown where they participate in thepredicted secondary structure. Several ligands were “disconnected”(equality symbol) in order to show their relatedness to the consensusmotif through circular permutation. The nucleotides predicted toparticipate in base pairing are indicated with underline invertedarrows, with the arrow heads pointing toward the helix junction. Thesequences are divided into two groups, A and B, based on the firstsingle stranded nucleotide (from the 5′ end) at the helix junction (A orG, between helices II and III). Mismatches in the helical regions areshown with dots under the corresponding letters (G-T and T-G base pairswere allowed). In places where single nucleotide bulges occur, themismatched nucleotide is shown above the rest of the sequence betweenits neighbors.

This classification is based in part on sequence homology among theseligands, but in greater part on the basis of a shared secondarystructure motif: a three-way helix junction with a three nucleotide loopat the branch point (FIG. 1) (SEQ ID NO: 82). These ligands weresubdivided into two groups; for ligands in group A, the loop at thebranch point has an invariant sequence AGC and in group B, that sequenceis G(T/G)(C/T). The proposed consensus secondary structure motif issupported by base-pairing covariation at non-conserved nucleotides inthe helices (Table 4). Since the three-way junctions are encoded incontinuous DNA strands, two of the helices end in loops at the distalend from the junction. These loops are highly variable, both in lengthand in sequence. Furthermore, through circular permutation of theconsensus motif, the loops occur in all three helices, although they aremost frequent in helices II and III. Together these observations suggestthat the regions distal from the helix junction are not important forhigh affinity binding to PDGF-AB. The highly conserved nucleotides areindeed found near the helix junction (Table 3, FIG. 1).

EXAMPLE 3 MINIMAL LIGAND DETERMINATIONS

The minimal sequence necessary for high affinity binding was determinedfor six of the best ligands to PDGF-AB. In general, the informationabout the 3′ and 5′ minimal sequence boundaries can be obtained bypartially fragmenting the nucleic acid ligand and then selecting for thefragments that retain high affinity for the target. With RNA ligands,the fragments can be conveniently generated by mild alkaline hydrolysis(Tuerk et al. (1990) J. Mol. Biol. 213: 749-761; Jellinek et al. (1994)Biochemistry 33:10450-10456; Jellinek et al. (1995) Biochemistry34:11363-11372; Green et al. (1995) J. Mol. Biol. 247:60-68). Since DNAis more resistant to base, an alternative method of generating fragmentsis needed for DNA. To determine the 3′ boundary, a population of ligandfragments serially truncated at the 3′ end was generated by extendingthe 5′ end-labeled primer annealed to the 3′ invariant sequence of a DNAligand using the dideoxy sequencing method. This population wasaffinity-selected by nitrocellulose filtration and the shortestfragments (truncated from the 3′ end) that retain high affinity bindingfor PDGF-AB were identified by polyacrylamide gel electrophoresis. The5′ boundary was determined in an analogous manner except that apopulation of 3′ end-labeled ligand fragments serially truncated at the5′ end was generated by limited digestion with lambda exonuclease. Theminimal ligand is then defined as the sequence between the twoboundaries. It is important to keep in mind that, while the informationderived from these experiments is useful, the suggested boundaries areby no means absolute since the boundaries are examined one terminus atthe time. The untruncated (radiolabeled) termini can augment, reduce orhave no effect on binding (Jellinek et al. (1994) Biochemistry33:10450-10456).

Of the six minimal ligands for which the boundaries were determinedexperimentally, two (20t (SEQ ID NO: 83) and 41t (SEQ ID NO: 85);truncated versions of ligands 20 and 41) bound with affinitiescomparable (within a factor of 2) to their full-length analogs and fourhad considerably lower affinities. The two minimal ligands that retainedhigh affinity binding to PDGF, 20t and 41t, contain the predictedthree-way helix junction secondary structure motif (FIG. 2) (SEQ ID NOS:83-85). The sequence of the third minimal ligand that binds to PDGF-ABwith high affinity, 36t, was deduced from the knowledge of the consensusmotif (FIG. 2). In subsequent experiments, we found that thesingle-stranded region at the 5′ end of ligand 20t is not important forhigh affinity binding. Furthermore, the trinucleotide loops on helicesII and III in ligand 36t (GCA and CCA) can be replaced with hexaethyleneglycol spacers (infra). These experiments provide further support forthe importance of the helix junction region in high affinity binding toPDGF-AB.

Binding of the Minimal Ligands to PDGF. The binding of minimal ligands20t, 36t, and 41t to varying concentrations of PDGF-AA, PDGF-AB andPDGF-BB is shown in FIGS. 3A-3C. In agreement with the bindingproperties of their full length analogs, the minimal ligands bind toPDGF-AB and PDGF-BB with substantially higher affinity than to PDGF AA(FIGS. 3A-3C, Table 5). In fact, their affinity for PDGF-AA iscomparable to that of random DNA (data not shown). The binding toPDGF-AA is adequately described with a monophasic binding equation whilethe binding to PDGF-AB and PDGF-BB is notably biphasic. In previousSELEX experiments, biphasic binding has been found to be a consequenceof the existence of separable nucleic acid species that bind to theirtarget protein with different affinities (Jellinek et al. (1995)Biochemistry 34:11363-11372) and unpublished results). The identity ofthe high and the low affinity fractions is at present not known. Sincethese DNA ligands described here were synthesized chemically, it ispossible that the fraction that binds to PDGF-AB and PDGF-BB with loweraffinity represents chemically imperfect DNA. Alternatively, the highand the low affinity species may represent stable conformational isomersthat bind to the PDGF B-chain with different affinities. In any event,the higher affinity binding component is the most populated ligandspecies in all cases (FIGS. 3A-3C). For comparison, a 39-mer DNA ligandthat binds to human thrombin with a K_(d) of 0.5 nM (ligand T39 (SEQ IDNO.: 88)):

5′-CAGTCCGTGGTAGGGCAGGTTGGGGTGACTTCGTGGAA[3′T], where [3′T] represents a3′-3′ linked thymidine nucleotide added to reduce 3′-exonucleasedegradation) and has a predicted stem-loop structure, binds to PDGF-ABwith a K_(d) of 0.23 μM (data not shown).

Dissociation Rates of the Minimal Ligands. To evaluate the kineticstability of the PDGF-AB/DNA complexes, the dissociation rates at 37° C.for the complexes of minimal ligands 20t, 36t and 41t with PDGF-AB weredetermined by measuring the amount of radiolabeled ligands (0.17 nM)bound to PDGF-AB (1 nM) as a function of time following the addition ofa large excess of unlabeled ligands (FIG. 4). At these protein and DNAligand concentrations, only the high affinity fraction of the DNAligands binds to PDGF-AB. The following values for the dissociation rateconstants were obtained by fitting the data points shown in FIG. 4 tothe first-order rate equation: 4.5±0.2×10⁻³ s⁻¹ (t_(½)=2.6 min) forligand 20t, 3.0±0.2×10⁻³ s⁻¹ (t_(½)=3.8 min) for lilgand 36t, and1.7±0.1×10⁻³ s⁻¹ (t_(½)=6.7 min) for ligand 41t. The association ratescalculated for the dissociation constants and dissociation rateconstants (k_(on)=k_(off)/K_(d)) are 3.1×10⁷ M⁻¹s⁻¹ for 20t, 3.1×10⁷M⁻¹s⁻¹ for 36t and 1.2×10⁷ M⁻¹s⁻¹ for 41t.

Melting Temperatures of the Minimal Ligands. Melting temperatures(T_(m)'s) were determined for minimal ligands 20t, 36t and 41t from theUV absorption vs. temperature profiles (FIG. 5). At the oligonucleotideconcentrations used in these experiments (320-440 nM), only themonomeric species were observed as single bands on non-denaturingpolyacrylamide gels. The T_(m) values were obtained from the firstderivative replots of the melting profiles. Ligands 20t and 41texhibited monophasic melting with T_(m) values of 44° C. and 49° C. Themelting profile of ligand 36t was biphasic, with the Tm value of 44° C.for the first (major) transition and ≈63° C. for the second transition.

Photocrosslinking of 5-Iodo-2′-Deoxyuridine Substituted Minimal DNALigands to PDGF-AB. To determine the sites on the DNA ligands and PDGFthat are in close contact, a series of photo-crosslinking experimentswas performed with 5′-iodo-2′-deoxyuridine (IdU)-substituted DNA ligands20t, 36t and 41t. Upon monochromatic excitation at 308 nm, 5-iodo- and5-bromo-substituted pyrimidine nucleotides populate a reactive tripletstate following intersystem crossing from the initial n to π*transition. The excited triplet state species then reacts with electronrich amino acid residues (such as Trp, Tyr and His) that are in itsclose proximity to yield a covalent crosslink. This method has been usedextensively in studies of nucleic acid-protein interactions since itallows irradiation with >300 nm light which minimizes photodamage(Willis et al. (1994) Nucleic Acids Res. 22:4947-4952; Stump and Hall(1995) RNA 1:55-63; Willis et al. (1993) Science 262:1255-1257; Jensenet al. (1995) Proc. Natl. Acad. Sci. U.S.A. 92:12220-12224). Analogs ofligands 20t, 36t and 41t were synthesized in which all thymidineresidues were replaced with IdU residues using the solid phasephosphoramidite method. The affinity of these IdU-substituted ligandsfor PDGF-AB was somewhat enhanced compared to the unsubstituted ligandsand based on the appearance of bands with slower electrophoreticmobility on 8% polyacrylamide/7 M urea gels, all three 5′ end-labeledIdU-substituted ligands crosslinked to PDGF-AB upon irradiation at 308nm (data not shown). The highest crosslinking efficiency was observedwith IdU-substituted ligand 20t. To identify the specific IdUposition(s) responsible for the observed crosslinking, seven singly ormultiply IdU-substituted analogs of 20t were tested for their ability tophoto-crosslink to PDGF-AB: ligands 20t-I1 through 20t-17(5-TGGGAGGGCGCGT¹T¹CT¹T¹CGT²GGT³T⁴ACT⁵T⁶T⁶T⁶AGT⁷CCCG-3′ (SEQ ID NOS.:89-95) where the numbers indicate IdU substitutions at indicatedthymidine nucleotides for the seven ligands). Of these seven ligands,efficient crosslinking to PDGF-AB was observed only with ligand 20t-I14(SEQ ID NO: 92). The photo-reactive IdU position corresponds to the 3′proximal thymidine in the loop at the helix junction (FIG. 2).

To identify the crosslinked amino acid residue(s) on PDGF-AB, a mixtureof 5′ end-labeled 20t-I4 and PDGF-AB was incubated for 15 min at 37° C.followed by irradiation at 308 nm. The reaction mixture was thendigested with modified trypsin and the crosslinked fragments resolved onan 8% polyacrylamide/7 M urea gel. Edman degradation of the peptidefragment recovered from the band that migrated closest to the free DNAband revealed the amino acid sequence KKPIXKK (SEQ ID NO: 96), where Xindicates a modified amino acid that could not be identified with the 20derivatized amino acid standards. This peptide sequence, where X isphenylalanine, corresponds to amino acids 80-86 in the PDGF-B chain(Johnsson et al. (1984) EMBO J. 3:921-928) which in the crystalstructure of PDGF-BB comprises a part of solvent-exposed loop III(Oefner et al. (1992) EMBO J. 11:3921-3926). In the PDGF A-chain, thispeptide sequence does not occur (Betsholtz et al. (1986) Nature320:695-699). Together, these data establish a point contact between aspecific thymidine residue in ligand 20t and phenylalanine 84 of thePDGF B-chain.

Receptor Binding Assay. In order to determine whether the DNA ligands toPDGF were able to inhibit the effects of PDGF isoforms on culturedcells, the effects on binding of ¹²⁵I-labeled PDGF isoforms to PDGF α-and β-receptors stably expressed in porcine aortic endothelial (PAE)cells by transfection were first determined. Ligands 20t, 36t and 41tall efficiently inhibited the binding of ¹²⁵I-PDGF-BB to PDGFα-receptors (FIG. 6) or PDGF β-receptors (data not shown), with halfmaximal effects around 1 nM of DNA ligand. DNA ligand T39 (describedsupra), directed against thrombin and included as a control, showed noeffect. None of the ligands was able to inhibit the binding of¹²⁵I-PDGF-AA to the PDGF α-receptor (data not shown), consistent withthe observed specificity of ligands 20t, 36t and 41t for PDGF-BB andPDGF-AB.

Inhibition of Mitogenic Effects by Minimal Ligands. The ability of theDNA ligands to inhibit the mitogenic effects of PDGF-BB on PAE cellsexpressing PDGF β-receptors was investigated. As shown in FIG. 7, thestimulatory effect of PDGF-BB on [³H]thymidine incorporation wasneutralized by ligands 20t, 36t and 41t. Ligand 36t exhibited halfmaximal inhibition at the concentration of 2.5 nM; ligands 41t wasslightly more efficient and 20t slightly less efficient. The controlligand T39 had no effect. Moreover, none of the ligands inhibited thestimulatory effects of fetal calf serum on [³H]thymidine incorporationin these cells, showing that the inhibitory effects are specific forPDGF.

EXAMPLE 4 POST-SELEX MODIFICATIONS

The stability of nucleic acids to nucleases is an importantconsideration in efforts to develop nucleic acid-based therapeutics.Experiments have shown that many, and in some cases most of thenucleotides in SELEX-derived ligands can be substituted with modifiednucleotides that resist nuclease digestion, without compromising highaffinity binding (Green et al. (1995) Chemistry and Biology 2:683-695;Green et al. (1995) J. Mol. Biol. 247:60-68).

A series of substitution experiments were conducted to identifypositions in ligand 36t that tolerate 2′-O-methyl (2′-O-Me) or 2′-fluoro(2′-F) substitution. Tables 6 and 7 and FIGS. 8A and 8B summarize thesubstitutions examined and their effect on the affinity of the modifiedligands for PDGF-AB or PDGF-BB. 2-Fluoropyrimidine nucleosidephosphoramidites were obtained from JBL Scientific (San Louis Obispo,Calif.). 2′-O-Methylpurine phosphoramidites were obtained fromPerSeptive Biosystems (Boston, Mass.). All other nucleosidephosphoramidites were from PerSeptive Biosystems (Boston, Mass.). Notall substitution combinations were examined. Nevertheless, theseexperiments have been used to identify the pattern of 2′-O-Me and 2′-Fsubstitutions that are compatible with high affinity binding to PDGF-ABor PDGF-BB. It is worth noting that trinucleotide loops on helices IIand III in ligand 36t (FIGS. 2 and 8B) can be replaced withpentaethylene glycol (18-atom) spacers (Spacer Phosphoramidite 18, GlenResearch, Sterling, Va.) (see Example 5 for description of synthesis ofpentaethylene glycol-substituted ligand) without compromising highaffinity binding to PDGF-AB or -BB. This is in agreement with the notionthat the helix junction domain of the ligand represents the core of thestructural motif required for high affinity binding. In practical terms,the replacement of six nucleotides with two pentaethylene glycol spacersis advantageous in that it reduces by four the number of coupling stepsrequired for the synthesis of the ligand. In addition to thesubstitution experiments, four nucleotides from the base of helix I werefound that could be deleted without loss of binding affinity (comparefor example ligand 36t with 36ta or ligand 1266 with 1295 in Tables 6and 7).

EXAMPLE 5 SYNTHESIS OF PEG-MODIFIED PDGF NUCLEIC ACID LIGANDS

A) General Procedure for the Synthesis of NX31975 on Solid Support

Synthesis was carried out on lmmol scale on a millipore 8800 automatedsynthesizer using standard deoxynucleoside phosphoramidites,2′-O-methyl-5′-O-DMT-N2-tert-butylphenoxyacetylguanosine-phosphoramidite,2′-O-methyl-5′-O-DMT-N6-tert-butylphenoxyacetyl-adenosine-phosphoramidite,2′-deoxy-2′-fluoro-5′-O-DMT-uridine-phosphoramidite,2′-deoxy-2′-fluoro-5′-O-DMT-N4-acetylcytidine-3′-N,N-diisopropyl-(2-cyanoethyl)-phosphoramidite,18-O-DMT-hexaethyleneglycol-1-[N,N-diisopropyl-(2-cyanoethyl)-phosphoramidite,] (FIG. 9C), and5-trifloroacetamidopentane-1-[N,N-diisopropyl-(2-cyanoethyl)-phosphoramidite,].(FIG. 9D). The syntheses were carried out using 4,5-dicyanoimidazole asthe activator on controlled pore glass (CPG) support of 600 A pore size,80-120 mesh, and 60-70 μmol/g loading with 5′-succinyl thymidine. Afterthe synthesis, the oligos were deprotected with 40% NH₄OH, at 55° C. for16h. The support was filtered, and washed with water and 1:1acetonitrile/water and the combined washings were evaporated to dryness.The ammonium counterion on the backbone was exchanged fortriethylammonium ion by reverse phase salt exchange and the solvent wasevaporated to afford the crude oligo as the triethylammonium salt.

Hexaethylene glycol spacers on the loops are attached to the nucleotidesthrough phosphate linkages. The structures of the 2 loops are shown inFIGS. 9A and 9B. The 5′ phosphate group shown is from the hexaethyleneglycol phosphoramidite.

B) Conjugation of 40K PEG NHS ester to the aminolinker on PDGF NucleicAcid Ligands

The NX31975 crude oligonucleotide containing the 5′ primary amino groupwas dissolved in 100 mM sodium borate buffer (pH 9) to 60 mg /mlconcentration. In a separate tube 2 Eq of PEG NHS ester (FIG. 9E)(Shearwater Polymers, Inc.) was dissolved in dry DMF (Ratio of borate:DMF 1:1) and the mixture was warmed to dissolve the PEG NHS ester. Thenthe oligo solution was quickly added to PEG solution and the mixture wasvigorously stirred at room temperature for 10 minutes. About 95% of theoligo conjugated to the PEG NHS ester.

EXAMPLE 6 STABILITY OF MODIFIED LIGANDS IN SERUM

The stabilities of DNA (36ta) and modified DNA (NX21568) ligands in ratserum at 37° C. were compared. Serum used for these experiments wasobtained from a Sprague-Dawley rat and was filtered through 0.45 μmcellulose acetate filter and buffered with 20 mM sodium phosphatebuffer. Test ligands (36ta or NX21568) were added to the serum at thefinal concentration of 500 nM. The final serum concentration was 85% asa result of the addition of buffer and ligand. From the original 900 μlincubation mixture, 100 μl aliquots were withdrawn at various timepoints and added to 10 μl of 500 mM EDTA (pH 8.0), vortexed and frozenon dry ice and stored at −20° C. until the end of the experiment. Theamount of full length oligonucleotide ligand remaining for each of thetime points was quantitated by HPLC analysis. To prepare the samples forHPLC injections, 200 μl of a mixture of 30% formamide, 70% 25 mM Trisbuffer (pH 8.0) containing 1% acetonitrile was added to 100 μl of thawedtime point samples, vortexed for 5 seconds and centrifuged for 20minutes at 14,000 rpm in an Eppendorf microcentrifuge. The analysis wasperformed using an anion exchange chromatography column (NuceoPac,Dionex, PA-100, 4×50 mm) applying a LiCl gradient. The amount of fulllength oligonucleotide remaining at each time point was determined fromthe peak areas (FIG. 10). With a half-life of about 500 min, themodified ligand (NX21568) exhibited a substantially greater stability inrat serum compared with the DNA ligand (36ta), which was degraded with ahalf-life of about 35 min (FIG. 10). Thus, the increase in stability inserum results from the 2′-substitutions.

EXAMPLE 7 EFFICACY OF NX31975-40K PEG IN RESTENOSIS

Rat Restenosis Model and Efficacy Results. The plasma residence time ofNucleic Acid Ligands is dramatically improved by the addition of large,inert functional groups such as polyethylene glycol (see for examplePCT/US 97/18944). For in vivo efficacy experiments, 40K PEG wasconjugated to NX31975 to create NX31975 40K PEG as described in Example5B (see FIG. 9A for molecular description). Importantly, based onbinding experiments, the addition of 40 kDa PEG group at the 5′-end ofthe ligand does not affect its binding affinity for PDGF-BB.

The effect of selective inhibition of PDGF-B by NX31975-40K PEG wasstudied in three-month-old male Sprague-Dawley rats (370-450 g). Therats were housed three to a cage with free access to a standardlaboratory diet and water. Artificial light was provided 14 hours perday. The experiments were performed in accordance with the institutionalguidelines at the Animal Department, Department of Surgery, UniversityHospital, Uppsala University, Sweden.

A total of 30 rats were randomly allocated to one of two treatmentgroups: 15 rats in group one received 10 mg/kg body weight of NX31975-40K PEG in phosphate buffered saline (PBS) twice daily delivered byintraperitoneal (i.p.) injections and 15 rats in group two (the controlgroup) received an equal volume of PBS (about 1 ml). The duration oftreatment was 14 days. The first injections in both groups were givenone hour before arterial injury.

To generate the arterial lesions, all animals were anaesthetized with ani.p. injection of a mixture of one part Fentanyl-fluanisone (Hypnormvet, fluanisone 10 mg/ml, fentanyl 0.2 mg/ml, Janssen Pharmaceutica,Beerse, Belgium), one part midazolam (Dormicum, Midazolam 5 mg/ml. F.Hoffman-La Roche AG, Basel, Switzerland) and two parts sterile water,0.33 ml/100 g rat. The distal left common carotid and external carotidarteries were exposed through a midline incision in the neck. The leftcommon carotid artery was traumatized by intraluminal passage of 2FFogarty embolectomy catheter introduced through the external carotidartery. The catheter was passed three times with the balloon expandedsufficiently with 0.06 ml distilled water to achieve a distension of thecarotid itself. The external carotid was ligated after removal of thecatheter and the wound was closed. All surgical procedures wereperformed by a surgeon blinded to the treatment groups.

Fourteen days after the catheter injury, the animals were anesthetizedas above. Twenty minutes before the exposure of the abdominal aorta theanimals received an intravenous injection of 0.5 ml 0.5% Evans blue dye(Sigma Chemical Co., St. Louis, Mo.) to allow identification of thevessel segment which remained deendothelialized. The carotid arterieswere perfused with ice-chilled PBS in situ at 100 mm Hg, via a largecannula placed retrograde in the abdominal aorta until the effluent ranclear via inferior caval vein vent. A distal half of the right and leftcommon carotid arteries, up to the level of the bifurcation, wereremoved and frozen in liquid nitrogen. Immediately thereafter, theremaining proximal segment was perfusion-fixed through the same aorticcannula at 100 mm Hg pressure with 2.5% glutaraldehyde in phosphatebuffer, pH 7.3. Before starting perfusion with PBS, the animals werekilled by an overdose of phenobarbital. After approximately 15 minutesof perfusion fixation, the remaining proximal right and left commoncarotid arteries were retrieved for further preparation, including theaortic arch and innominate artery.

Five sections, approximately 0.5 μm apart, from the middle of the Evansblue stained segment of the left common carotid artery and one sectionfrom the contralateral uninjured artery were analyzed per animal withcomputer-assisted planimetry. The following areas were measured: thearea encircled by external elastic lumina (EEL), internal elastic lumina(IEL) and the endoluminal cell layer. Areas for tunical media and tunicaintima were calculated. All measurements by an individual blinded to thetreatment regimens.

Based on values of intima/media ratios for the control and the NucleicAcid Ligand-treated groups, the PDGF Nucleic Acid Ligand significantly(p<0.05) inhibited about 50% of the neointima formation (FIG. 11).

EXAMPLE 8 ANTAGONISM OF PDGF IN GLOMERULONEPHRITIS BY NX31975-40K PEG

This example provides the general procedures followed and incorporatedin Example 9.

Materials and Methods

All Nucleic Acid Ligands and their sequence-scrambled controls weresynthesized by the solid phase phosphoramidite method on controlled poreglass using an 8800 Milligen DNA Synthesizer and deprotected usingammonium hydroxide at 55° C. for 16 h. The Nucleic Acid Ligand used inexperiments described in this example and Example 9 is NX31975 40KPEG(FIG. 9A). NX31975 40K PEG was created by conjugating NX31975 (Table7) to 40K PEG as described in Example 5. In the sequence-scrambledcontrol Nucleic Acid Ligand, eight nucleotides in the helix junctionregion of NX31975 were interchanged without formally changing theconsensus secondary structure (see FIG. 8C). The binding affinity of thesequence-scrambled control Nucleic Acid Ligand for PDGF BB is ˜1 μM,which is 10,000 fold lower compared to NX21617. The sequence-scrambledcontrol Nucleic Acid Ligand was then conjugated to PEG and named NX3197640K PEG (see FIG. 9B for molecular description). The covalent couplingof PEG to the Nucleic Acid Ligand (or to the sequence-scrambled control)was accomplished as described in Example 5.

Rat PDGF-BB for cross-reactivity binding experiments was derived from E.coli transfected with sCR-Script Amp SK(+) plasmid containing the ratPDGF-BB sequence. Rat PDGF-BB sequence was derived rat lung poly A+RNA(Clonetech, San Diego, Calif.) through RT-PCR using primers that amplifysequence encoding the mature form of PDGF-BB. Rat PDGF-BB proteinexpression and purification was performed at R&D Systems.

Mesangial Cell Culture Experiments

Human mesangial cells were established in culture, characterized andmaintained as described previously (Radeke et al. (1994) J. Immunol.153:1281-1292). To examine the antiproliferative effect of the ligandson the cultured mesangial cells, cells were seeded in 96-well plates(Nunc, Wiesbaden, Germany) and grown to subconfluency. They were thengrowth-arrested for 48 hours in MCDB 302 medium (Sigma, Deisenhofen,Germany). After 48 hours various stimuli together with either 50 or 10μg/ml Nucleic Acid Ligand NX31975 40K PEG or 50 or 10 μg/mlsequence-scrambled Nucleic Acid Ligand (NX31976 40K PEG) were added:medium alone, 100 ng/ml human recombinant PDGF-AA, -AB or -BB (kindlyprovided by J. Hoppe, University of Würzburg, Germany), 100 ng/ml humanrecombinant epidermal growth factor (EGF; Calbiochem, Bad Soden,Germany) or 100 ng/ml recombinant human fibroblast growth factor-2(kindly provided by Synergen, Boulder, Colo.). Following 72 hours ofincubation, numbers of viable cells were determined using2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide(XTT; Sigma) as described (Lonnemann et al. (1995) Kidney Int.51:837-844).

Experimental Design

Anti-Thy 1.1 mesangial proliferative glomerulonephritis was induced in33 male Wistar rats (Charles River, Sulzfeld, Germany) weighing 150-160gby injection of 1 mg/kg monoclonal anti-Thy 1.1 antibody (clone OX-7;European Collection of Animal Cell Cultures, Salisbury, England). Ratswere treated with Nucleic Acid Ligands or PEG (see below) from day 3 to8 after disease induction. Treatment consisted of twice daily i.v. bolusinjections of the substances dissolved in 400 μl PBS, pH 7.4. Thetreatment duration was chosen to treat rats from about one day after theonset to the peak of mesangial cell proliferation (Floege et al. (1993)Kidney Int. Suppl. 39:S47-54). Four groups of rats were studied: 1) ninerats, who received NX31975 40K PEG (i.e., a total of 4 mg of the PDGF-Bligand coupled to 15.7 mg 40K PEG); 2) ten rats, who received anequivalent amount of PEG-coupled, scrambled Nucleic Acid Ligand (NX3197640K PEG); 3) eight rats, who received an equivalent amount (15.7 mg) of40K PEG alone; 4) six rats, who received 400 μl bolus injections of PBSalone. Renal biopsies for histological evaluation were obtained on days6 and 9 after disease induction. Twenty-four hour urine collections wereperformed from days 5 to 6 and 8 to 9 after disease induction. Thethymidine analogue 5-bromo-2′-deoxyuridine (BrdU; Sigma, Deisenhofen,Germany; 100 mg/kg body weight) was injected intraperitoneally at 4hours prior to sacrifice on day 9.

Normal ranges of proteinuria and renal histological parameter (seebelow) were established in 10 non-manipulated Wistar rats of similarage.

Renal Morphology

Tissue for light microscopy and immunoperoxidase staining was fixed inmethyl Carnoy's solution (Johnson et al. (1990) Am. J. Pathol.136:369-374) and embedded in paraffin. Four μm sections were stainedwith the periodic acid Schiff (PAS) reagent and counterstained withhematoxylin. In the PAS stained sections the number of mitoses within100 glomerular tufts was determined.

Immunoperoxidase Staining

Four mm sections of methyl Carnoy's fixed biopsy tissue were processedby an indirect immunoperoxidase technique as described (Johnson et al.(1990) Am. J. Pathol. 136:369-374). Primary antibodies were identical tothose described previously (Burg et al. (1997) Lab. Invest. 76:505-516;Yoshimura et al. (1991) Kidney Int. 40:470-476) and included a murinemonoclonal antibody (clone 1A4) to α-smooth muscle actin; a murinemonoclonal antibody (clone PGF-007) to PDGF B-chain; a murine monoclonalIgG antibody (clone ED1) to a cytoplasmic antigen present in monocytes,macrophages and dendritic cells; affinity purified polyclonal goatanti-human/bovine type IV collagen IgG preabsorbed with raterythrocytes; an affinity purified IgG fraction of a polyclonal rabbitanti-rat fibronectin antibody; plus appropriate negative controls asdescribed previously (Burg et al. (1997) Lab. Invest. 76:505-516;Yoshimura et al. (1991) Kidney Int. 40:470-476). Evaluation of allslides was performed by an observer, who was unaware of the origin ofthe slides.

To obtain mean numbers of infiltrating leukocytes in glomeruli, morethan 50 consecutive cross sections of glomeruli containing more than 20discrete capillary segments were evaluated and mean values per kidneywere calculated. For the evaluation of the immunoperoxidase stains forα-smooth muscle actin, PDGF B-chain, type IV collagen and fibronectineach glomerular area was graded semiquantitatively, and the mean scoreper biopsy was calculated. Each score reflects mainly changes in theextent rather than intensity of staining and depends on the percentageof the glomerular tuft area showing focally enhanced positive staining:I=0-25%, II=25-50%, III=50-75%, IV=>75%. This semiquantitative scoringsystem is reproducible among different observers and the data are highlycorrelated with those obtained by computerized morphometry (Kliem et al.(1996) Kidney Int. 49:666-678; Hugo et al. (1996) J. Clin. Invest.97:2499-2508).

Immunohistochemical Double-Staining

Double immunostaining for the identification of the type ofproliferating cells was performed as reported previously. (Kliem et al.(1996) Kidney Int. 49:666-678; Hugo et al. (1996) J. Clin. Invest.97:2499-2508) by first staining the sections for proliferating cellswith a murine monoclonal antibody (clone BU-1) againstbromo-deoxyuridine containing nuclease in Tris buffered saline(Amersham, Braunschweig, Germany) using an indirect immunoperoxidaseprocedure. Sections were then incubated with the IgG₁ monoclonalantibodies 1A4 against α-smooth muscle actin and ED1 againstmonocytes/macrophages. Cells were identified as proliferating mesangialcells or monocytes/macrophages if they showed positive nuclear stainingfor BrdU and if the nucleus was completely surrounded by cytoplasmpositive for α-smooth muscle actin. Negative controls included omissionof either of the primary antibodies in which case no double-staining wasnoted.

In situ Hybridization for Type IV Collagen mRNA

In situ hybridization was performed on 4 mm sections of biopsy tissuefixed in buffered 10% formalin utilizing a digoxigenin-labelledanti-sense RNA probe for type IV collagen (Eitner et al. (1997) KidneyInt. 51:69-78) as described (Yoshimura et al. (1991) Kidney Int.40:470-476). Detection of the RNA probe was performed with an alkalinephosphatase coupled anti-digoxigenin antibody (Genius NonradioactiveNucleic Acid Detection Kit, Boehringer-Mannheim, Mannheim, Germany) withsubsequent color development. Controls consisted of hybridization with asense probe to matched serial sections, by hybridization of theanti-sense probe to tissue sections which had been incubated with RNAseA before hybridization, or by deletion of the probe, antibody or colorsolution described (Yoshimura et al. (1991) Kidney Int. 40:470-476).Glomerular mRNA expression was semiquantitatively assessed using thescoring system described above.

Miscellaneous Measurements

Urinary protein was measured using the Bio-Rad Protein Assay (Bio-RadLaboratories GmbH, München, Germany) and bovine serum albumin (Sigma) asa standard.

Statistical Analysis

All values are expressed as means±SD. Statistical significance (definedas p<0.05) was evaluated using ANOVA and Bonferroni t-tests.

EXAMPLE 9

For all experiments reported here, the modified DNA Nucleic Acid Ligandwas conjugated to 40K PEG as described in Examples 5 and 8 and shown inFIGS. 9A and 9B. Since most Nucleic Acid Ligands have molecular weightsranging between 8 to 12 kDa (the modified PDGF Nucleic Acid Ligand hasMW of 10 kDa), the addition of a large inert molecular entity such asPEG dramatically improves the residence times of Nucleic Acid Ligands invivo (see for example PCT/US 97/18944). Importantly, the addition of thePEG moiety to the 5′ end of the Nucleic Acid Ligand has no effect on thebinding affinity of the Nucleic Acid Ligand for PDGF-BB (K_(d)˜1×10⁻¹⁰M).

Cross-reactivity of Nucleic Acid Ligands for Rat PDGF-BB

The sequence of PDGF is highly conserved among species, and human andrat PDGF B-chain sequences are 89% identical (Herren et al. (1993)Biochim. Biophys. Acta 1173:294; Lindner et al. (1995) Circ. Res.76:951). Nevertheless, in view of the high specificity of Nucleic AcidLigands (Gold et al. (1995) Ann. Rev. Biochem. 64:763-797), the correctinterpretation of the in vivo experiments requires understanding of thebinding properties of the Nucleic Acid Ligands to rat PDGF B-chain. Wehave therefore cloned and expressed the mature form of rat PDGF-BB in E.coli. The PDGF Nucleic Acid Ligands bound to rat and human recombinantPDGF-BB with the same high affinity (data not shown).

PDGF B-Chain DNA-Ligand Specifically Inhibits Mesangial CellProliferation in vitro

In growth arrested mesangial cells, the effects of NX31975 40K PEG orthe scrambled Nucleic Acid Ligand (NX31976 40K PEG) on growth factorinduced proliferation were tested. Stimulated growth rates of the cellswere not affected by the addition of scrambled Nucleic Acid Ligand (FIG.12). Fifty μg/ml of NX31975 40K PEG significantly reduced PDGF-BBinduced mesangial cell growth (FIG. 12). PDGF-AB and -AA inducedmesangial cell growth also tended to be lower with NX31975 40K PEG, butthese differences failed to reach statistical significance (FIG. 12). Incontrast, no effects of NX31975 40K PEG on either EGF or FGF-2 inducedgrowth were noted. Similar effects were noted if the Nucleic AcidLigands were used at a concentration of 10 μg/ml (data not shown).

Effects of PDGF B-Chain DNA-Ligand in Rats with Anti-Thy 1.1 Nephritis

Following the injection of anti-Thy 1.1antibody, PBS treated animalsdeveloped the typical course of the nephritis, which is characterized byearly mesangiolysis and followed by a phase of mesangial cellproliferation and matrix accumulation on days 6 and 9 (Floege et al.(1993) Kidney Int. Suppl. 39: S47-54). No obvious adverse effects werenoted following the repeated injection of Nucleic Acid Ligands or PEGalone, and all rats survived and appeared normal until the end of thestudy.

In PAS stained renal sections the mesangioproliferative changes on days6 and 9 after disease induction were severe and indistinguishable amongrats receiving PBS, PEG alone or the scrambled Nucleic Acid Ligand (datanot shown). Histological changes were markedly reduced and almostnormalized in the NX31975 40K PEG ligand treated group In order to(semi-)quantitatively evaluate the mesangioproliferative changes,various parameters were analyzed:

a) Reduction of Mesangial Cell Proliferation

Glomerular cell proliferation, as assessed by counting the number ofglomerular mitoses, was not significantly different between the threecontrol groups on days 6 and 9 (FIG. 13A). As compared to rats receivingthe scrambled Nucleic Acid Ligand, treatment with PDGF-B ligand led to areduction of glomerular mitoses by 64% on day 6 and by 78% on day 9(FIG. 13A). To assess the treatment effects on mesangial cells, therenal sections for α-smooth muscle actin were immunostained, which isexpressed by activated mesangial cells only (Johnson et al. (1991) J.Clin. Invest. 87:847-858). Again, there were no significant differencesbetween the three control groups on days 6 and 9. However, the immunostaining scores of α-smooth muscle actin were significantly reduced onday 6 and 9 in the NX31975 40K PEG treated group (FIG. 13D). Tospecifically determine whether mesangial cell proliferation was reduced,NX31975 40K PEG treated rats and scrambled Nucleic Acid Ligand treatedrats were double immunostained for a cell proliferation marker (BrdU)and α-smooth muscle actin. The data confirmed a marked decrease ofproliferating mesangial cells on day 9 after disease induction: 2.2±0.8BrdU-/α-smooth muscle actin positive cells per glomerular cross sectionin PDGF-B aptamer treated rats versus 43.3±12.4 cells in rats receivingthe scrambled Nucleic Acid Ligand, i.e., a 95% reduction of mesangialcell proliferation. In contrast, no effect of the PDGF-B aptamer wasnoted on proliferating monocytes/macrophages on day 9 after diseaseinduction (PDGF-B aptamer treated rates: 2.8±1.1 BrdU+/ED-1+ cells per100 glomerular cross sections; scrambled aptamer treated rats: 2.7±1.8).

b) Reduced Expression of Endogenous PDGF B-Chain

By immunohistochemistry, the glomerular PDGF B-chain expression wasmarkedly upregulated in all three control groups (FIG. 13B), similar toprevious observations (Yoshimura et al. (1991) Kidney Int. 40:470-476).In the NX31975 40K PEG treated group the glomerular overexpression ofPDGF B-chain was significantly reduced in parallel with the reduction ofproliferating mesangial cells (FIG. 13B).

c) Reduction of Glomerular Monocyte/Macrophage Influx

The glomerular monocyte/macrophage influx was significantly reduced inthe NX31975 40K PEG treated rats as compared to rats receiving scrambledNucleic Acid Ligand on days 6 and 9 after disease induction (FIG. 13E).

d) Effects on Proteinuria

Moderate proteinuria of up to 147 mg/24 hrs was present on day 6 afterdisease induction in the 3 control groups (FIG. 13C). Treatment withNX31975 40K PEG reduced the mean proteinuria on day 6, but this failedto reach statistical significance (FIG. 13C). Proteinuria on day 9 afterdisease induction was low and similar in all four groups (FIG. 13C).

e) Reduction of Glomerular Matrix Production and Accumulation

By immunohistochemistry, marked glomerular accumulation of type IVcollagen and fibronectin was noted in all three control groups (FIG.14). The overexpression of both glomerular type IV collagen andfibronectin was significantly reduced NX31975 40K PEG treated rats (FIG.14). In the latter, glomerular staining scores approached those observedin normal rats (FIG. 14). By in situ hybridization, the decreasedglomerular expression of type IV collagen in NX31975 40K PEG treatedrats was shown to be associated with decreased glomerular synthesis ofthis collagen type (FIG. 14).

EXAMPLE 10 EXPERIMENTAL PROCEDURE FOR EVOLVING2′-FLUORO-2′-DEOXYPYRIMIDINE RNA LIGANDS TO PDGF AND RNA SEQUENCESOBTAINED.

2′-FLUORO-2′-DEOXYPYRIMIDINE RNA SELEX

SELEX with 2′-fluoro-2′-deoxypyrimidine RNA targeting PDGF AB was doneessentially as described previously (vide supra, and Jellinek et al.,1993, 1994: supra) using the primer template set as shown in Table 8(SEQ ID NOS: 36-38). Briefly, the 2′-fluoro-2′-deoxypyrimidine RNA foraffinity selections was prepared by in vitro transcription fromsynthetic DNA templates using T7 RNA polymerase (Milligan et al. (1987)Nucl. Acids Res. 15:8783). The conditions for in vitro transcriptiondescribed in detail previously (Jellinek et al. (1994) supra) were used,except that higher concentration (3 mM) of the2′-fluoro-2′-deoxypyrimidine nucleoside triphosphates (2′-F-UTP and2′-F-CTP) was used compared to ATP and GTP (1 mM). Affinity selectionswere done by incubating PDGF AB with 2′-fluoro-2′-deoxypyrimidine RNAfor at least 15 min at 37° C. in PBS containing 0.01% human serumalbumin. Partitioning of free RNA from protein-bound RNA was done bynitrocellulose filtration as described (Jellinek et al., 1993, 1994:supra). Reverse transcription of the affinity-selected RNA andamplification by PCR were done as described previously (Jellinek et al.(1994) supra). Nineteen rounds of SELEX were performed, typicallyselecting between 1-12% of the input RNA. For the first eight rounds ofselection, suramin (3-15 μM) was included in the selection buffer toincrease the selection pressure. The affinity-enriched pool (round 19)was cloned and sequenced as described (Schneider et al. (1992) supra).Forty-six unique sequences have been identified, and the sequences areshown in Table 9 (SEQ ID NOS: 39-81). The unique-sequence ligands werescreened for their ability to bind PDGF AB with high affinity. Whilerandom 2′-fluoropyrimidine RNA (Table 8) bound to PDGF with adissociation constant (Kd) of 35±7 nM, many of the affinity-selectedligands bound to PDGF AB with ≈100-fold higher affinities. Among theunique ligands, clones 9 (K_(d)=91±16 pM), 11 (K_(d)=120±21 pM), 16(K_(d)=116±34 pM), 23 (K_(d)=173±38 pM), 25 (K_(d)=80±22 pM), 37(K_(d)=97±29 pM), 38 (K_(d)=74±39 pM), and 40 (K_(d)=91±32 pM) exhibitedthe highest affinity for PDGF AB (binding of all of these ligands toPDGF AB is biphasic and the K_(d) for the higher affinity bindingcomponent is given).

TABLE 1 Starting DNA and PCR primers for the ssDNA SELEX experiment. SEQID NO. Starting ssDNA:5′-ATCCGCCTGATTAGCGATACT[-40N-]ACTTGAGCAAAATCACCTGCAGGGG-3′ 1 PCR Primer3N2*: 5′-BBBCCCCTGCAGGTGATTTTGCTCAAGT-3′ 2 PCR Primer 5N2**:5′-CCGAAGCTTAATACGACTCACTATAGGGATCCGCCTGATTAGCGATACT-3′ 3 *B = biotinphosphoramidite (e. g., Glen Research, Sterling, VA) **For rounds 10,11, and 12, the truncated PCR primer 5N2 (underlined) was used toamplify the template.

TABLE 2 Unique Sequences of the ssDNA high affinity ligands to PDGF.5′-ATCCGCCTGATTAGCGATACT[40N]ACTTGAGCAAAATCACCTGCAGGGG-3′ SEQ ID NO *14AGGCTTGACAAAGGGCACCATGGCTTAGTGGTCCTAGT  4 *41CAGGGCACTGCAAGCAATTGTGGTCCCAATGGGCTGAGT  5   6CCAGGCAGTCATGGTCATTGTTTACAGTCGTGGAGTAGGT  6  23AGGTGATCCCTGCAAAGGCAGGATAACGTCCTGAGCATC  7   2ATGTGATCCCTGCAGAGGGAGGANACGTCTGAGCATC  8  34CACGTGATCCCATAAGGGCTGCGCAAAATAGCAGAGCATC  9   8GGTGGACTAGAGGGCAGCAAACGATCCTTGGTTAGCGTCC 10   1GGTGCGACGAGGCTTACACAAACGTACACGTTTCCCCGC 11   5TGTCGGAGCAGGGGCGTACGAAAACTTTACAGTTCCCCCG 12 *40AGTGGAACAGGGCACGGAGAGTCAAACTTTGGTTTCCCCC 13  47GTGGGTAGGGATCGGTGGATGCCTCGTCACTTCTAGTCCC 14  18GGGCGCCCTAAACAAAGGGTGGTCACTTCTAGTCCCAGGA 15  30TCCGGGCTCGGGATTCGTGGTCACTTTCAGTCCCGGATATA 16 *20ATGGGAGGGCGCGTTCTTCGTGGTTACTTTTAGTCCCG 17  35ACGGGAGGGCACGTTCTTCGTGGTTACTTTTAGTCCCG 18  13GCTCGTAGGGGGCGATTCTTTCGCCGTTACTTCCAGTCCT 19  16GAGGCATGTTAACATGAGCATCGTCTCACGATCCTCAGCC 20 *36CCACAGGCTACGGCACGTAGAGCATCACCATGATCCTGTG 21  50GCGGGCATGGCACATGAGCATCTCTGATCCCGCAATCCTC 22   4ACCGGGCTACTTCGTAGAGCATCTCTGATCCCGGTGCTCG 23  44AAAGGGCGAACGTAGGTCGAGGCATCCATTGGATCCCTTC 24  24ACGGGCTCTGTCACTGTGGCACTAGCAATAGTCCCGTCGC 25   7GGGCAGACCTTCTGGACGAGCATCACCTATGTGATCCCG 26 *26AGAGGGGAAGTAGGCTGCCTGACTCGAGAGAGTCCTCCCG 27  19AGGGGTGCGAAACACATAATCCTCGCGGATTCCCATCGCT 28  48GGGGGGGCAATGGCGGTACCTCTGGTCCCCTAAATAC 29  46GCGGCTCAAAGTCCTGCTACCCGCAGCACATCTGTGGTC 30  25TTGGGCGTGAATGTCCACGGGTACCTCCGGTCCCAAAGAG 31  31TCCGCGCAAGTCCCTGGTAAAGGGCAGCCCTAACTGGTC 32  12CAAGTTCCCCACAAGACTGGGGCTGTTCAAACCGCTAGTA 33  15CAAGTAGGGCGCGACACACGTCCGGGCACCTAAGGTCCCA 34 *38AAAGTCGTGCAGGGTCCCCTGGAAGCATCTCCGATCCCAG 35 *Indicates a boundaryexperiment was performed. Italics indicate the clones that were found toretain high affinity binding as minimal ligands.

TABLE 3

TABLE 4 Frequency of base pairs in the helical regions of the consensusmotif shown in FIG. 1. Base pair^(b) Position^(a) AT TA GG CG TG GTother I-1 0 0 21 0 0 0 0 I-2 0 0 21 0 0 0 0 I-3 5 0 16 0 0 0 0 I-4 3 5 14 1 0 7 I-5 2 3 3 4 0 0 9 II-1 0 1 2 17 0 0 1 II-2 5 5 5 1 0 4 1 II-3 34 7 6 0 0 1 II-4 3 0 8 5 0 0 4 III-1 21 0 0 0 0 0 0 III-2 0 10 0 11 0 00 III-3 0 7 0 13 1 0 0 ^(a)Helices are numbered with roman numerals asshown in FIG. 1. Individual base pairs are numbered with arabic numeralsstarting with position 1 at the helix junction and increasing withincreased distance from the junction. ^(b)The TG and GT base pairs tothe Watson-Crick base pairs for this analysis were included. There is atotal of 21 sequences in the set.

TABLE 5 Affinities of the minimal DNA ligands to PDGF AA, PDGF AB andPDGF BB. K_(d), nM Ligand PDGF AA^(a) PDGF AB^(b) PDGF BB^(b) 20t 47 ± 40.147 ± 0.011 0.127 ± 0.031 36t  72 ± 12 0.094 ± 0.011 0.093 ± 0.009 41t49 ± 8 0.138 ± 0.009 0.129 ± 0.011 ^(a)Data points shown in FIG. 3A werefitted to eq 1 (Example 1). ^(b)Data points in FIGS. 3B and 3C werefitted to eq. 2. The dissociation constant (K_(d)) values shown are forthe higher affinity binding component. The mole fraction of DNA thatbinds to PDGF AB or PDGF BB as the high affinity component rangesbetween 0.58 to 0.88. The K_(d) values for the lower affinityinteraction range between 13 to 78 nM.

TABLE 6 Relative affinity for PDGF-AB of ligand 36t variants. SEQ LigandID NO Composition* Kd^(ligand)/Kd^(36t)** 36t  84CACAGGCTACGGCACGTAGAGCATCACCATGATCCTGTG[^(3′)T] 1.0 1073  97 CACAGGCTACGGCACGTAGAGCATCACCATGATCCT GUG [^(3′)T] 11.8 1074  98 CACAGGCTACGGCACGU AGAGCATCACCATGATCCTGTG[^(3′)T] 3.1 1075  99CACAGGCTACGGCACGTAGAGCATC ACCAU GATCCTGTG[^(3′)T] 10 1076 100 CAC AGGCTACGGCACGU AGAGCATC ACCAU GATCCTGTG[^(3′)T] 440 1145 101CACAGGCTACGGCACGTAGAGCAT CACCA TGATCCTGTG[^(3′)T] 0.27 1148 102 CACAGGCUA CGGCACGTAGAGCATCACCATGATCCTGTG[^(3′)T] 281 1144 103CACAGGCTACGGCACGTAGAGCATCACCAT GAUCCU GTG[^(3′)T] 994 1142 104CACAGGCTACGGCACG U AGAGC AU CACCATGATCCTGTG[^(3′)T] 12.9 1149 105CACAGGCTACGGCACGTA GAGC ATCACCATGATCCTGTG[^(3′)T] 2.9 EV1 106CACAGGCTACGGCACGTAG AGC ATCACCATGATCCTGTG[^(3′)T] 35.1 EV2 107CACAGGCTACG GCACGU AGAGCATCACCATGATCCTGTG[^(3′)T] 5.3 EV3 108 CACAGGCTACGGCA CGTAGAGCATCACCATGATCCTGTG[^(3′)T] 1.5 EV4 109 CACAGGCTACGGCACGTAGAGCATCACCATGATCCTGTG[^(3′)T] 4.5 EV5 110CACAGGCTACGGCACGTAGAGCATCACCATGATCC UGUG [^(3′)T] 2.3 1157 111

1.0 1160 112

1.4 1161 113

0.22 1162 114

0.52 1165 115

0.61 1164 116

0.45 1166 117

0.76 1159 118

0.37 1163 119

1.3 1158 120

2.4 1255 121

24.2 1257 122

1.3 1265 123

1.4 1266 124

1.0 1267 125

4.2 1269 126

0.87 1295 127

0.9 1296 128

2.1 1297 129

2.9 1303 130 CAGGCUA CGGCA CGTAGAGCA UCACCA TGAT CCUG [^(3′)T] 5.8 1304131 CAGG CUACGGCA CGTAGAGCA UCACCA TGAT CCUG [^(3′)T] 607 1305 132 CAGGCU A CGGCA CGTAG A GCA UCACCA TGAT CCUG [^(3′)T] 196 1306 133 CAGG CU ACGGCA CGTAGA G CA UCACCA TGAT CCUG [^(3′)T] 4.4 1327 134

0.63 1328 135

2.2 1329 136

0.72 1369 137

0.37 1374 138

1.5 1358 139

0.54 1441 140

0.33

**Kd^(36t) value of 0.178 ± 88 pM used for the calculation is average,with standard deviation, of four independent measurements (94 ± 11, 161± 24, 155 ± 30 and 302 ± 32 pM).

TABLE 7 Relative affinity for PDGF-BB of ligand 36ta variants. Theeffect of various substitutions on the affinity of ligand 36ta forPDGF-BB. SEQ. Ligand Composition* Kd^(ligand)/Kd^(36ta)** ID NO. 36taCAGGCTACGGCACGTAGAGCATCACCATGATCCTG[^(3′)T] 1.0 141 NX21568

0.63 142 NX21617

0.54 143 NX21618

418 144 NX31975

0.54 148 NX31976

149

**Kd^(36ta)= 0.159 ± 13 pM.

TABLE 8 Starting RNA and PCR primers for the 2′-fluoropyrimidine RNASELEX experiment. SEQ ID NO Starting 2′-fluoropyrimidine RNA: StartingRNA: 5′-GGGAGACAAGAAUAACGCUCAA[-50 N-]UUCGACAGGAGGCUCACAACAGGC-3′ 36 PCRPrimer 1: 5′-TAATACGACTCACTATAGGGAGACAAGAATAACGCTCAA-3′ 37 PCR Primer 2:5′-GCCTGTTGTGAGCCTCCTGTCGAA-3′ 38

TABLE 9 Sequences of the evolved region of 2′-fluoropyrimidine RNA highaffinity ligands to PDGF AB. Sequences of the fixed region (Table 8) arenot shown. SEQ ID NO.  1 CGGUGGCAUUUCUUCACUUCCUUCUCGCUUUCUCGCGUUGGGCNCGA39  2 CCAACCUUCUGUCGGCGUUGCUUUUUGGACGGCACUCAGGCUCCA 40  3UCGAUCGGUUGUGUGCCGGACAGCCUUAACCAGGGCUGGGACCGAGGCC 41  4CUGAGUAGGGGAGGAAGUUGAAUCAGUUGUGGCGCCUCUCAUUCGC 42  5CAGCACUUUCGCUUUUCAUCAUUUUUUCUUUCCACUGUUGGGCGCGGAA 43  6UCAGUGCUGGCGUCAUGUCUCGAUGGGGAUUUUUCUUCAGCACUUUGCCA 44  7UCUACUUUCCAUUUCUCUUUUCUUCUCACGAGCGGGUUUCCAGUGAACCA 45  8CGAUAGUGACUACGAUGACGAAGGCCGCGGGUUGGAUGCCCGCAUUGA 46 10GUCGAUACUGGCGACUUGCUCCAUUGGCCGAUUAACGAUUCGGUCAG 47 13GUGCAAACUUAACCCGGGAACCGCGCGUUUCGAUCGACUUUCCUUUCCA 48 15AUUCCGCGUUCCGAUUAAUCCUGUGCUCGGAAAUCGGUAGCCAUAGUGCA 49 16CGAACGAGGAGGGAGUGGCAAGGGAUGGUUGGAUAGGCUCUACGCUCA 50 17GCGAAACUGGCGACUUGCUCCAUUGGCCGAUAUAACGAUUCGGUUCAU 51 18CGAACGAGGAGGGAGUCGCAAGGGAUGGUUGGAUAGGCUCUACGCUCAA 52 19CGAGAAGUGACUACGAUGACGAAGGCCGCGGGUUGAAUCCCUCAUUGA 53 20AAGCAACGAGACCUGACGCCUGAUGUGACUGUGCUUGCACCCGAUUCUG 54 21GUGAUUCUCAUUCUCAAUGCUUUCUCACAACUUUUUCCACUUCAGCGUGA 55 22AAGCAACGAGACUCGACGCCUGAUGUGACUGUGCUUGCACCCGAUUCU 56 23UCGAUCGGUUGUGUGCCGGACAGCUUUGACCAUGAGCUGGGACCGAGGCC 57 24NGACGNGUGGACCUGACUAAUCGACUGAUCAAAGAUCCCGCCCAGAUGGG 58 26CACUGCGACUUGCAGAAGCCUUGUGUGGCGGUACCCCCUUUGGCCUCG 59 27GGUGGCAUUUCUUCAUUUUCCUUCUCGCUUUCUCCGCCGUUGGGCGCG 60 29CCUGAGUAGGGGGGAAAGUUGAAUCAGUUGUGGCGCUCUACUCAUUCGCC 61 30GUCGAAACUGGCGACUUGCUCCAUUGGCCGAUAUAACGAUUCGGUUCA 62 31GCGAUACUGGCGACUUGCUCCAUUGGCCGAUAUAACGAUUCGGCUCAG 63 32ACGUGGGGCACAGGACCGAGAGUCCCUCCGGCAAUAGCCGCUACCCCACC 64 33CACAGCCUNANAGGGGGGAAGUUGAAUCAGUUGUGGCGCUCUACUCAUUCGC 65 34ANGGGNUAUGGUGACUUGCUCCAUUGGCCGAUAUAACGAUUCGGUCAG 66 35CCUGCGUAGGGNGGGAAGUUGAAUCAGUUGUGGCGCUCUACUCAUUCGCC 67 39CGAACGAGGAGGGAGUGGCAAGGGAUGGUUGGAUAGGCUCUACGCUCA 68 41GUGCAAACUUAACCCGGGAACCGCGCGUUUCGAUUCGCUUUCCNUAUUCCA 69 42CGAACGAGGAGGGAGUGGCAAGGGACGGUNNAUAGGCUCUACGCUCA 70 43UCGGUGUGGCUCAGAAACUGACACGCGUGAGCUUCGCACACAUCUGC 71 44UAUCGCUUUUCAUCAAUUCCACUUUUUCACUCUNUAACUUGGGCGUGCA 72 45GUGCAAACUUAACCCGGGAACCGCGCGUUUCGAUCCUGCAUCCUUUUUCC 73 46UCGNUCGGUUGUGUGCCGGCAGCUUUGUCCAGCGUUGGGCCGAGGCC 74 47AGUACCCAUCUCAUCUUUUCCUUUCCUUUCUUCAAGGCACAUUGAGGGU 75 49CCUGAGUAGGGGGGGAAGUUGAACCAGUUGUGGCNGCCUACUCAUUCNCCA 76 51CCNNCCUNCUGUCGGCGCUUGUCUUUUUGGACGGGCAACCCAGGGCUC 77 52CCAACCUNCUGUCGGCGCUUGUCUUUUUGGACGAGCAACUCAAGGCUCGU 78 53CCAGCGCAGAUCCCGGGCUGAAGUGACUGCCGGCAACGGCCGCUCCA 79 54UUCCCGUAACAACUUUUCAUUUUCACUUUUCAUCCAACCAGUGAGCAGCA 80 55UAUCGCUUUCAUCAAAUUCCACUCCUUCACUUCUUUAACUUGGGCGUGCA 81

149 86 base pairs nucleic acid single linear DNA unknown 1 ATCCGCCTGATTAGCGATAC TNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 50 NNNNNNNNNN NACTTGAGCAAAATCACCTG CAGGGG 86 25 base pairs nucleic acid single linear DNAunknown 2 CCCCTGCAGG TGATTTTGCT CAAGT 25 49 base pairs nucleic acidsingle linear DNA unknown 3 CCGAAGCTTA ATACGACTCA CTATAGGGAT CCGCCTGATTAGCGATACT 49 84 base pairs nucleic acid single linear DNA unknown 4ATCCGCCTGA TTAGCGATAC TAGGCTTGAC AAAGGGCACC ATGGCTTAGT 50 GGTCCTAGTACTTGAGCAAA ATCACCTGCA GGGG 84 85 base pairs nucleic acid single linearDNA unknown 5 ATCCGCCTGA TTAGCGATAC TCAGGGCACT GCAAGCAATT GTGGTCCCAA 50TGGGCTGAGT ACTTGAGCAA AATCACCTGC AGGGG 85 86 base pairs nucleic acidsingle linear DNA unknown 6 ATCCGCCTGA TTAGCGATAC TCCAGGCAGT CATGGTCATTGTTTACAGTC 50 GTGGAGTAGG TACTTGAGCA AAATCACCTG CAGGGG 86 85 base pairsnucleic acid single linear DNA unknown 7 ATCCGCCTGA TTAGCGATACTAGGTGATCC CTGCAAAGGC AGGATAACGT 50 CCTGAGCATC ACTTGAGCAA AATCACCTGCAGGGG 85 83 base pairs nucleic acid single linear DNA unknown 8ATCCGCCTGA TTAGCGATAC TATGTGATCC CTGCAGAGGG AGGANACGTC 50 TGAGCATCACTTGAGCAAAA TCACCTGCAG GGG 83 86 base pairs nucleic acid single linearDNA unknown 9 ATCCGCCTGA TTAGCGATAC TCACGTGATC CCATAAGGGC TGCGCAAAAT 50AGCAGAGCAT CACTTGAGCA AAATCACCTG CAGGGG 86 86 base pairs nucleic acidsingle linear DNA unknown 10 ATCCGCCTGA TTAGCGATAC TGGTGGACTA GAGGGCAGCAAACGATCCTT 50 GGTTAGCGTC CACTTGAGCA AAATCACCTG CAGGGG 86 85 base pairsnucleic acid single linear DNA unknown 11 ATCCGCCTGA TTAGCGATACTGGTGCGACG AGGCTTACAC AAACGTACAC 50 GTTTCCCCGC ACTTGAGCAA AATCACCTGCAGGGG 85 86 base pairs nucleic acid single linear DNA unknown 12ATCCGCCTGA TTAGCGATAC TTGTCGGAGC AGGGGCGTAC GAAAACTTTA 50 CAGTTCCCCCGACTTGAGCA AAATCACCTG CAGGGG 86 86 base pairs nucleic acid single linearDNA unknown 13 ATCCGCCTGA TTAGCGATAC TAGTGGAACA GGGCACGGAG AGTCAAACTT 50TGGTTTCCCC CACTTGAGCA AAATCACCTG CAGGGG 86 86 base pairs nucleic acidsingle linear DNA unknown 14 ATCCGCCTGA TTAGCGATAC TGTGGGTAGG GATCGGTGGATGCCTCGTCA 50 CTTCTAGTCC CACTTGAGCA AAATCACCTG CAGGGG 86 86 base pairsnucleic acid single linear DNA unknown 15 ATCCGCCTGA TTAGCGATACTGGGCGCCCT AAACAAAGGG TGGTCACTTC 50 TAGTCCCAGG AACTTGAGCA AAATCACCTGCAGGGG 86 87 base pairs nucleic acid single linear DNA unknown 16ATCCGCCTGA TTAGCGATAC TTCCGGGCTC GGGATTCGTG GTCACTTTCA 50 GTCCCGGATATAACTTGAGC AAAATCACCT GCAGGGG 87 84 base pairs nucleic acid singlelinear DNA unknown 17 ATCCGCCTGA TTAGCGATAC TATGGGAGGG CGCGTTCTTCGTGGTTACTT 50 TTAGTCCCGA CTTGAGCAAA ATCACCTGCA GGGG 84 84 base pairsnucleic acid single linear DNA unknown 18 ATCCGCCTGA TTAGCGATACTACGGGAGGG CACGTTCTTC GTGGTTACTT 50 TTAGTCCCGA CTTGAGCAAA ATCACCTGCAGGGG 84 86 base pairs nucleic acid single linear DNA unknown 19ATCCGCCTGA TTAGCGATAC TGCTCGTAGG GGGCGATTCT TTCGCCGTTA 50 CTTCCAGTCCTACTTGAGCA AAATCACCTG CAGGGG 86 86 base pairs nucleic acid single linearDNA unknown 20 ATCCGCCTGA TTAGCGATAC TGAGGCATGT TAACATGAGC ATCGTCTCAC 50GATCCTCAGC CACTTGAGCA AAATCACCTG CAGGGG 86 86 base pairs nucleic acidsingle linear DNA unknown 21 ATCCGCCTGA TTAGCGATAC TCCACAGGCT ACGGCACGTAGAGCATCACC 50 ATGATCCTGT GACTTGAGCA AAATCACCTG CAGGGG 86 86 base pairsnucleic acid single linear DNA unknown 22 ATCCGCCTGA TTAGCGATACTGCGGGCATG GCACATGAGC ATCTCTGATC 50 CCGCAATCCT CACTTGAGCA AAATCACCTGCAGGGG 86 86 base pairs nucleic acid single linear DNA unknown 23ATCCGCCTGA TTAGCGATAC TACCGGGCTA CTTCGTAGAG CATCTCTGAT 50 CCCGGTGCTCGACTTGAGCA AAATCACCTG CAGGGG 86 86 base pairs nucleic acid single linearDNA unknown 24 ATCCGCCTGA TTAGCGATAC TAAAGGGCGA ACGTAGGTCG AGGCATCCAT 50TGGATCCCTT CACTTGAGCA AAATCACCTG CAGGGG 86 86 base pairs nucleic acidsingle linear DNA unknown 25 ATCCGCCTGA TTAGCGATAC TACGGGCTCT GTCACTGTGGCACTAGCAAT 50 AGTCCCGTCG CACTTGAGCA AAATCACCTG CAGGGG 86 85 base pairsnucleic acid single linear DNA unknown 26 ATCCGCCTGA TTAGCGATACTGGGCAGACC TTCTGGACGA GCATCACCTA 50 TGTGATCCCG ACTTGAGCAA AATCACCTGCAGGGG 85 86 base pairs nucleic acid single linear DNA unknown 27ATCCGCCTGA TTAGCGATAC TAGAGGGGAA GTAGGCTGCC TGACTCGAGA 50 GAGTCCTCCCGACTTGAGCA AAATCACCTG CAGGGG 86 86 base pairs nucleic acid single linearDNA unknown 28 ATCCGCCTGA TTAGCGATAC TAGGGGTGCG AAACACATAA TCCTCGCGGA 50TTCCCATCGC TACTTGAGCA AAATCACCTG CAGGGG 86 83 base pairs nucleic acidsingle linear DNA unknown 29 ATCCGCCTGA TTAGCGATAC TGGGGGGGCA ATGGCGGTACCTCTGGTCCC 50 CTAAATACAC TTGAGCAAAA TCACCTGCAG GGG 83 85 base pairsnucleic acid single linear DNA unknown 30 ATCCGCCTGA TTAGCGATACTGCGGCTCAA AGTCCTGCTA CCCGCAGCAC 50 ATCTGTGGTC ACTTGAGCAA AATCACCTGCAGGGG 85 86 base pairs nucleic acid single linear DNA unknown 31ATCCGCCTGA TTAGCGATAC TTTGGGCGTG AATGTCCACG GGTACCTCCG 50 GTCCCAAAGAGACTTGAGCA AAATCACCTG CAGGGG 86 85 base pairs nucleic acid single linearDNA unknown 32 ATCCGCCTGA TTAGCGATAC TTCCGCGCAA GTCCCTGGTA AAGGGCAGCC 50CTAACTGGTC ACTTGAGCAA AATCACCTGC AGGGG 85 86 base pairs nucleic acidsingle linear DNA unknown 33 ATCCGCCTGA TTAGCGATAC TCAAGTTCCC CACAAGACTGGGGCTGTTCA 50 AACCGCTAGT AACTTGAGCA AAATCACCTG CAGGGG 86 86 base pairsnucleic acid single linear DNA unknown 34 ATCCGCCTGA TTAGCGATACTCAAGTAGGG CGCGACACAC GTCCGGGCAC 50 CTAAGGTCCC AACTTGAGCA AAATCACCTGCAGGGG 86 86 base pairs nucleic acid single linear DNA unknown 35ATCCGCCTGA TTAGCGATAC TAAAGTCGTG CAGGGTCCCC TGGAAGCATC 50 TCCGATCCCAGACTTGAGCA AAATCACCTG CAGGGG 86 96 base pairs nucleic acid single linearRNA unknown 36 GGGAGACAAG AAUAACGCUC AANNNNNNNN NNNNNNNNNN NNNNNNNNNN 50NNNNNNNNNN NNNNNNNNNN NNUUCGACAG GAGGCUCACA ACAGGC 96 39 base pairsnucleic acid single linear RNA unknown 37 TAATACGACT CACTATAGGGAGACAAGAAT AACGCTCAA 39 24 base pairs nucleic acid single linear RNAunknown 38 GCCTGTTGTG AGCCTCCTGT CGAA 24 93 base pairs nucleic acidsingle linear RNA unknown All pyrimidines are 2′-F modified 39GGGAGACAAG AAUAACGCUC AACGGUGGCA UUUCUUCACU UCCUUCUCGC 50 UUUCUCGCGUUGGGCNCGAU UCGACAGGAG GCUCACAACA GGC 93 91 base pairs nucleic acidsingle linear RNA unknown All pyrimidines are 2′-F modified 40GGGAGACAAG AAUAACGCUC AACCAACCUU CUGUCGGCGU UGCUUUUUGG 50 ACGGCACUCAGGCUCCAUUC GACAGGAGGC UCACAACAGG C 91 95 base pairs nucleic acid singlelinear RNA unknown All pyrimidines are 2′-F modified 41 GGGAGACAAGAAUAACGCUC AAUCGAUCGG UUGUGUGCCG GACAGCCUUA 50 ACCAGGGCUG GGACCGAGGCCUUCGACAGG AGGCUCACAA CAGGC 95 92 base pairs nucleic acid single linearRNA unknown All pyrimidines are 2′-F modified 42 GGGAGACAAG AAUAACGCUCAACUGAGUAG GGGAGGAAGU UGAAUCAGUU 50 GUGGCGCCUC UCAUUCGCUU CGACAGGAGGCUCACAACAG GC 92 95 base pairs nucleic acid single linear RNA unknownAll pyrimidines are 2′-F modified 43 GGGAGACAAG AAUAACGCUC AACAGCACUUUCGCUUUUCA UCAUUUUUUC 50 UUUCCACUGU UGGGCGCGGA AUUCGACAGG AGGCUCACAACAGGC 95 96 base pairs nucleic acid single linear RNA unknown Allpyrimidines are 2′-F modified 44 GGGAGACAAG AAUAACGCUC AAUCAGUGCUGGCGUCAUGU CUCGAUGGGG 50 AUUUUUCUUC AGCACUUUGC CAUUCGACAG GAGGCUCACAACAGGC 96 96 base pairs nucleic acid single linear RNA unknown Allpyrimidines are 2′-F modified 45 GGGAGACAAG AAUAACGCUC AAUCUACUUUCCAUUUCUCU UUUCUUCUCA 50 CGAGCGGGUU UCCAGUGAAC CAUUCGACAG GAGGCUCACAACAGGC 96 94 base pairs nucleic acid single linear RNA unknown Allpyrimidines are 2′-F modified 46 GGGAGACAAG AAUAACGCUC AACGAUAGUGACUACGAUGA CGAAGGCCGC 50 GGGUUGGAUG CCCGCAUUGA UUCGACAGGA GGCUCACAACAGGC 94 93 base pairs nucleic acid single linear RNA unknown Allpyrimidines are 2′-F modified 47 GGGAGACAAG AAUAACGCUC AAGUCGAUACUGGCGACUUG CUCCAUUGGC 50 CGAUUAACGA UUCGGUCAGU UCGACAGGAG GCUCACAACA GGC93 95 base pairs nucleic acid single linear RNA unknown All pyrimidinesare 2′-F modified 48 GGGAGACAAG AAUAACGCUC AAGUGCAAAC UUAACCCGGGAACCGCGCGU 50 UUCGAUCGAC UUUCCUUUCC AUUCGACAGG AGGCUCACAA CAGGC 95 96base pairs nucleic acid single linear RNA unknown All pyrimidines are2′-F modified 49 GGGAGACAAG AAUAACGCUC AAAUUCCGCG UUCCGAUUAA UCCUGUGCUC50 GGAAAUCGGU AGCCAUAGUG CAUUCGACAG GAGGCUCACA ACAGGC 96 94 base pairsnucleic acid single linear RNA unknown All pyrimidines are 2′-F modified50 GGGAGACAAG AAUAACGCUC AACGAACGAG GAGGGAGUGG CAAGGGAUGG 50 UUGGAUAGGCUCUACGCUCA UUCGACAGGA GGCUCACAAC AGGC 94 94 base pairs nucleic acidsingle linear RNA unknown All pyrimidines are 2′-F modified 51GGGAGACAAG AAUAACGCUC AAGCGAAACU GGCGACUUGC UCCAUUGGCC 50 GAUAUAACGAUUCGGUUCAU UUCGACAGGA GGCUCACAAC AGGC 94 95 base pairs nucleic acidsingle linear RNA unknown All pyrimidines are 2′-F modified 52GGGAGACAAG AAUAACGCUC AACGAACGAG GAGGGAGUCG CAAGGGAUGG 50 UUGGAUAGGCUCUACGCUCA AUUCGACAGG AGGCUCACAA CAGGC 95 94 base pairs nucleic acidsingle linear RNA unknown All pyrimidines are 2′-F modified 53GGGAGACAAG AAUAACGCUC AACGAGAAGU GACUACGAUG ACGAAGGCCG 50 CGGGUUGAAUCCCUCAUUGA UUCGACAGGA GGCUCACAAC AGGC 94 95 base pairs nucleic acidsingle linear RNA unknown All pyrimidines are 2′-F modified 54GGGAGACAAG AAUAACGCUC AAAAGCAACG AGACCUGACG CCUGAUGUGA 50 CUGUGCUUGCACCCGAUUCU GUUCGACAGG AGGCUCACAA CAGGC 95 95 base pairs nucleic acidsingle linear RNA unknown All pyrimidines are 2′-F modified 55GGGAGACAAG AAUAACGCUC AAGUGAUUCU CAUUCUCAAU GCUUUCUCAC 50 AACUUUUUCCACUUCAGCGU GAUUCGACAG GAGGCUCACA CAGGC 95 94 base pairs nucleic acidsingle linear RNA unknown All pyrimidines are 2′-F modified 56GGGAGACAAG AAUAACGCUC AAAAGCAACG AGACUCGACG CCUGAUGUGA 50 CUGUGCUUGCACCCGAUUCU UUCGACAGGA GGCUCACAAC AGGC 94 96 base pairs nucleic acidsingle linear RNA unknown All pyrimidines are 2′-F modified 57GGGAGACAAG AAUAACGCUC AAUCGAUCGG UUGUGUGCCG GACAGCUUUG 50 ACCAUGAGCUGGGACCGAGG CCUUCGACAG GAGGCUCACA ACAGGC 96 96 base pairs nucleic acidsingle linear RNA unknown All pyrimidines are 2′-F modified 58GGGAGACAAG AAUAACGCUC AANGACGNGU GGACCUGACU AAUCGACUGA 50 UCAAAGAUCCCGCCCAGAUG GGUUCGACAG GAGGCUCACA ACAGGC 96 94 base pairs nucleic acidsingle linear RNA unknown All pyrimidines are 2′-F modified 59GGGAGACAAG AAUAACGCUC AACACUGCGA CUUGCAGAAG CCUUGUGUGG 50 CGGUACCCCCUUUGGCCUCG UUCGACAGGA GGCUCACAAC AGGC 94 94 base pairs nucleic acidsingle linear RNA unknown All pyrimidines are 2′-F modified 60GGGAGACAAG AAUAACGCUC AAGGUGGCAU UUCUUCAUUU UCCUUCUCGC 50 UUUCUCCGCCGUUGGGCGCG UUCGACAGGA GGCUCACAAC AGGC 94 96 base pairs nucleic acidsingle linear RNA unknown All pyrimidines are 2′-F modified 61GGGAGACAAG AAUAACGCUC AACCUGAGUA GGGGGGAAAG UUGAAUCAGU 50 UGUGGCGCUCUACUCAUUCG CCUUCGACAG GAGGCUCACA ACAGGC 96 94 base pairs nucleic acidsingle linear RNA unknown All pyrimidines are 2′-F modified 62GGGAGACAAG AAUAACGCUC AAGUCGAAAC UGGCGACUUG CUCCAUUGGC 50 CGAUAUAACGAUUCGGUUCA UUCGACAGGA GGCUCACAAC AGGC 94 94 base pairs nucleic acidsingle linear RNA unknown All pyrimidines are 2′-F modified 63GGGAGACAAG AAUAACGCUC AAGCGAUACU GGCGACUUGC UCCAUUGGCC 50 GAUAUAACGAUUCGGCUCAG UUCGACAGGA GGCUCACAAC AGGC 94 96 base pairs nucleic acidsingle linear RNA unknown All pyrimidines are 2′-F modified 64GGGAGACAAG AAUAACGCUC AAACGUGGGG CACAGGACCG AGAGUCCCUC 50 CGGCAAUAGCCGCUACCCCA CCUUCGACAG GAGGCUCACA ACAGGC 96 98 base pairs nucleic acidsingle linear RNA unknown All pyrimidines are 2′-F modified 65GGGAGACAAG AAUAACGCUC AACACAGCCU NANAGGGGGG AAGUUGAAUC 50 AGUUGUGGCGCUCUACUCAU UCGCUUCGAC AGGAGGCUCA CAACAGGC 98 94 base pairs nucleic acidsingle linear RNA unknown All pyrimidines are 2′-F modified 66GGGAGACAAG AAUAACGCUC AAANGGGNUA UGGUGACUUG CUCCAUUGGC 50 CGAUAUAACGAUUCGGUCAG UUCGACAGGA GGCUCACAAC AGGC 94 96 base pairs nucleic acidsingle linear RNA unknown All pyrimidines are 2′-F modified 67GGGAGACAAG AAUAACGCUC AACCUGCGUA GGGNGGGAAG UUGAAUCAGU 50 UGUGGCGCUCUACUCAUUCG CCUUCGACAG GAGGCUCACA ACAGGC 96 94 base pairs nucleic acidsingle linear RNA unknown All pyrimidines are 2′-F modified 68GGGAGACAAG AAUAACGCUC AACGAACGAG GAGGGAGUGG CAAGGGAUGG 50 UUGGAUAGGCUCUACGCUCA UUCGACAGGA GGCUCACAAC AGGC 94 97 base pairs nucleic acidsingle linear RNA unknown All pyrimidines are 2′-F modified 69GGGAGACAAG AAUAACGCUC AAGUGCAAAC UUAACCCGGG AACCGCGCGU 50 UUCGAUUCGCUUUCCNUAUU CCAUUCGACA GGAGGCUCAC AACAGGC 97 93 base pairs nucleic acidsingle linear RNA unknown All pyrimidines are 2′-F modified 70GGGAGACAAG AAUAACGCUC AACGAACGAG GAGGGAGUGG CAAGGGACGG 50 UNNAUAGGCUCUACGCUCAU UCGACAGGAG GCUCACAACA GGC 93 93 base pairs nucleic acidsingle linear RNA unknown All pyrimidines are 2′-F modified 71GGGAGACAAG AAUAACGCUC AAUCGGUGUG GCUCAGAAAC UGACACGCGU 50 GAGCUUCGCACACAUCUGCU UCGACAGGAG GCUCACAACA GGC 93 95 base pairs nucleic acidsingle linear RNA unknown All pyrimidines are 2′-F modified 72GGGAGACAAG AAUAACGCUC AAUAUCGCUU UUCAUCAAUU CCACUUUUUC 50 ACUCUNUAACUUGGGCGUGC AUUCGACAGG AGGCUCACAA CAGGC 95 96 base pairs nucleic acidsingle linear RNA unknown All pyrimidines are 2′-F modified 73GGGAGACAAG AAUAACGCUC AAGUGCAAAC UUAACCCGGG AACCGCGCGU 50 UUCGAUCCUGCAUCCUUUUU CCUUCGACAG GAGGCUCACA ACAGGC 96 93 base pairs nucleic acidsingle linear RNA unknown All pyrimidines are 2′-F modified 74GGGAGACAAG AAUAACGCUC AAUCGNUCGG UUGUGUGCCG GCAGCUUUGU 50 CCAGCGUUGGGCCGAGGCCU UCGACAGGAG GCUCACAACA GGC 93 95 base pairs nucleic acidsingle linear RNA unknown All pyrimidines are 2′-F modified 75GGGAGACAAG AAUAACGCUC AAAGUACCCA UCUCAUCUUU UCCUUUCCUU 50 UCUUCAAGGCACAUUGAGGG UUUCGACAGG AGGCUCACAA CAGGC 95 97 base pairs nucleic acidsingle linear RNA unknown All pyrimidines are 2′-F modified 76GGGAGACAAG AAUAACGCUC AACCUGAGUA GGGGGGGAAG UUGAACCAGU 50 UGUGGCNGCCUACUCAUUCN CCAUUCGACA GGAGGCUCAC AACAGGC 97 94 base pairs nucleic acidsingle linear RNA unknown All pyrimidines are 2′-F modified 77GGGAGACAAG AAUAACGCUC AACCNNCCUN CUGUCGGCGC UUGUCUUUUU 50 GGACGGGCAACCCAGGGCUC UUCGACAGGA GGCUCACAAC AGGC 94 96 base pairs nucleic acidsingle linear RNA unknown All pyrimidines are 2′-F modified 78GGGAGACAAG AAUAACGCUC AACCAACCUN CUGUCGGCGC UUGUCUUUUU 50 GGACGAGCAACUCAAGGCUC GUUUCGACAG GAGGCUCACA ACAGGC 96 93 base pairs nucleic acidsingle linear RNA unknown All pyrimidines are 2′-F modified 79GGGAGACAAG AAUAACGCUC AACCAGCGCA GAUCCCGGGC UGAAGUGACU 50 GCCGGCAACGGCCGCUCCAU UCGACAGGAG GCUCACAACA GGC 93 96 base pairs nucleic acidsingle linear RNA unknown All pyrimidines are 2′-F modified 80GGGAGACAAG AAUAACGCUC AAUUCCCGUA ACAACUUUUC AUUUUCACUU 50 UUCAUCCAACCAGUGAGCAG CAUUCGACAG GAGGCUCACA ACAGGC 96 96 base pairs nucleic acidsingle linear RNA unknown All pyrimidines are 2′-F modified 81GGGAGACAAG AAUAACGCUC AAUAUCGCUU UCAUCAAAUU CCACUCCUUC 50 ACUUCUUUAACUUGGGCGUG CAUUCGACAG GAGGCUCACA ACAGGC 96 23 base pairs nucleic acidsingle linear DNA unknown N at positions 1 and 23 is any base pair. N atpositions 5 and 10 is any base pair. N at positions 6 and 9 is any basepair. N at positions 7 and 8 is any base pair. 82 NGGCNNNNNN GRKYAYYRRTCCN 23 38 base pairs nucleic acid single linear DNA unknown Nucleotide38 is an inverted orientation T (3′-3′-linked) 83 TGGGAGGGCG CGTTCTTCGTGGTTACTTTT AGTCCCGT 38 40 base pairs nucleic acid single linear DNAunknown Nucleotide 40 is an inverted orientation T (3′-3′-linked) 84CACAGGCTAC GGCACGTAGA GCATCACCAT GATCCTGTGT 40 45 base pairs nucleicacid single linear DNA unknown Nucleotide 45 is an inverted orientationT (3′-3′-linked) 85 TACTCAGGGC ACTGCAAGCA ATTGTGGTCC CAATGGGCTG AGTAT 4536 base pairs nucleic acid single linear DNA unknown C at positions 11,25 and 26 is 2′-O- Methyl-2′-deoxycytidine. G at positions 9, 10, 17, 19and 35 is 2′-O-Methyl-2′-deoxyguanosine. A at positions 12, 24 and 27 is2′-O- Methyl-2′-deoxyadenosine. U at positions 6, 22 and 34 is 2′-fluoro-2′-deoxyuridine. C at positions 8, 23, 32 and 33 is 2′-fluoro-2′-deoxycytidine. Nucleotide 36 is an inverted orientation T(3′-3′-linked). 86 CAGGCUACGG CACGTAGAGC AUCACCATGA TCCUGT 36 32 basepairs nucleic acid single linear DNA unknown G at positions 9, 15, 17and 31 is 2′-O-methyl-2′-deoxyguanosine. A at position 22 is2′-O-methyl-2′- deoxyadenine. U at positions 6, 20 and 30 is2′-fluoro-2′-deoxyuridine. C at positions 8, 21, 28 and 29 is 2′-fluoro-2′-deoxycytidine. N at positions 10 and 23 is hexaethylene glycolphosphoramidite spacer. Nucleotide 32 is an inverted orientation T(3′-3′-linked). 87 CAGGCUACGN CGTAGAGCAU CANTGATCCU GT 32 39 base pairsnucleic acid single linear DNA unknown Nucleotide 39 is an invertedorientation T (3′-3′-linked). 88 CAGTCCGTGG TAGGGCAGGT TGGGGTGACTTCGTGGAAT 39 37 base pairs nucleic acid single linear DNA unknown T atpositions 13, 14, 16 and 17 is substituted with IdU. 89 TGGGAGGGCGCGTTCTTCGT GGTTACTTTT AGTCCCG 37 37 base pairs nucleic acid singlelinear DNA unknown T at position 20 is substituted with IdU. 90TGGGAGGGCG CGTTCTTCGT GGTTACTTTT AGTCCCG 37 37 base pairs nucleic acidsingle linear DNA unknown T at position 23 is substituted with IdU. 91TGGGAGGGCG CGTTCTTCGT GGTTACTTTT AGTCCCG 37 37 base pairs nucleic acidsingle linear DNA unknown T at position 24 is substituted with IdU. 92TGGGAGGGCG CGTTCTTCGT GGTTACTTTT AGTCCCG 37 37 base pairs nucleic acidsingle linear DNA unknown T at position 27 is substituted with IdU. 93TGGGAGGGCG CGTTCTTCGT GGTTACTTTT AGTCCCG 37 37 base pairs nucleic acidsingle linear DNA unknown T at positions 28-30 is substituted with IdU.94 TGGGAGGGCG CGTTCTTCGT GGTTACTTTT AGTCCCG 37 37 base pairs nucleicacid single linear DNA unknown T at position 33 is substituted with IdU.95 TGGGAGGGCG CGTTCTTCGT GGTTACTTTT AGTCCCG 37 7 amino acids amino acidsingle linear Peptide unknown Xaa at position 5 is a modified amino acidthat could not be identified. 96 Lys Lys Pro Ile Xaa Lys Lys 5 40 basepairs nucleic acid single linear DNA unknown C at positions 1 and 3 is2′-O-Methyl- 2′-deoxycytidine. A at position 2 is 2′-O-Methyl-2′-deoxyadenosine. G at positions 37 and 39 is 2′-O-Methyl-2′-deoxyguanosine. U at position 38 is 2′-O-Methyl-2′-deoxyuridine. Nucleotide 40 is an inverted orientation T (3′-3′-linked).97 CACAGGCTAC GGCACGTAGA GCATCACCAT GATCCTGUGT 40 40 base pairs nucleicacid single linear DNA unknown C at positions 10, 13 and 15 is 2′-O-Methyl-2′-deoxycytidine. G at positions 11, 12 and 16 is 2′-O-Methyl-2′-deoxyguanosine. A at position 14 is 2′-O-Methyl-2′-deoxyadenosine. U at position 17 is 2′-O-Methyl-2′- deoxyuridine.Nucleotide 40 is an inverted orientation T (3′-3′-linked). 98 CACAGGCTACGGCACGUAGA GCATCACCAT GATCCTGTGT 40 40 base pairs nucleic acid singlelinear DNA unknown A at positions 26 and 29 is 2′-O-Methyl-2′-deoxyadenosine. C at positions 27 and 28 is 2′-O-Methyl-2′-deoxycytidine. U at position 30 is 2′-O-Methyl-2′-deoxyuridine. Nucleotide 40 is an inverted orientation T (3′-3′-linked).99 CACAGGCTAC GGCACGTAGA GCATCACCAU GATCCTGTGT 40 40 base pairs nucleicacid single linear DNA unknown C at positions 1, 3, 10, 13, 15, 27, and28 is 2′-O-Methyl-2′-deoxycytidine. A at positions 2, 14, 26 and 29 is2′-O-Methyl-2′-deoxyadenosine. G at positions 11, 12 and 16 is 2′-O-Methyl-2′-deoxyguanosine. U at positions 17 and 30 is 2′-O-Methyl-2′-deoxyuridine. Nucleotide 40 is an inverted orientation T(3′-3′-linked). 100 CACAGGCTAC GGCACGUAGA GCATCACCAU GATCCTGTGT 40 40base pairs nucleic acid single linear DNA unknown C at positions 25, 27and 28 is 2′-O- Methyl-2′-deoxycytidine. A at positions 26 and 29 is2′-O- Methyl-2′-deoxyadenosine. Nucleotide 40 is an inverted orientationT (3′-3′-linked). 101 CACAGGCTAC GGCACGTAGA GCATCACCAT GATCCTGTGT 40 40base pairs nucleic acid single linear DNA unknown A at positions 4 and 9is 2′-O- Methyl-2′-deoxyadenosine. G at positions 5 and 6 is 2′-O-Methyl-2′-deoxyguanosine. C at position 7 is 2′-O-Methyl-2′- deoxycytidine. Uat position 8 is 2′-O-Methyl-2′- deoxyuridine. Nucleotide 40 is aninverted orientation T (3′-3′-linked). 102 CACAGGCUAC GGCACGTAGAGCATCACCAT GATCCTGTGT 40 40 base pairs nucleic acid single linear DNAunknown G at position 31 is 2′-O-Methyl-2′- deoxyguanosine. A atposition 32 is 2′-O-Methyl-2′- deoxyadenosine. U at positions 33 and 36is 2′-O- Methyl-2′-deoxyuridine. C at positions 34 and 35 is 2′-O-Methyl-2′-deoxycytidine. Nucleotide 40 is an inverted orientation T(3′-3′-linked). 103 CACAGGCTAC GGCACGTAGA GCATCACCAT GAUCCUGTGT 40 40base pairs nucleic acid single linear DNA unknown U at positions 17 and24 is 2′-O- Methyl-2′-deoxyuridine. A at position 23 is 2′-O-Methyl-2′-deoxyadenosine. Nucleotide 40 is an inverted orientation T(3′-3′-linked). 104 CACAGGCTAC GGCACGUAGA GCAUCACCAT GATCCTGTGT 40 40base pairs nucleic acid single linear DNA unknown G at positions 19 and21 is 2′-O- Methyl-2′-deoxyguanosine. A at position 20 is2′-O-Methyl-2′- deoxyadenosine. C at position 22 is 2′-O-Methyl-2′-deoxycytidine. Nucleotide 40 is an inverted orientation T(3′-3′-linked). 105 CACAGGCTAC GGCACGTAGA GCATCACCAT GATCCTGTGT 40 40base pairs nucleic acid single linear DNA unknown A at position 20 is2′-O-Methyl-2′- deoxyadenosine. G at position 21 is 2′-O-Methyl-2′-deoxyguanosine. C at position 22 is 2′-O-Methyl-2′- deoxycytidine.Nucleotide 40 is an inverted orientation T (3′-3′-linked). 106CACAGGCTAC GGCACGTAGA GCATCACCAT GATCCTGTGT 40 40 base pairs nucleicacid single linear DNA unknown G at positions 12 and 16 is 2′-O-Methyl-2′-deoxyguanosine. C at positions 13 and 15 is 2′-O-Methyl-2′-deoxycytidine. A at position 14 is 2′-O-Methyl-2′-deoxyadenosine. U at position 17 is 2′-O-Methyl-2′- deoxyuridine.Nucleotide 40 is an inverted orientation T (3′-3′-linked). 107CACAGGCTAC GGCACGUAGA GCATCACCAT GATCCTGTGT 40 40 base pairs nucleicacid single linear DNA unknown C at positions 10 and 13 is 2′-O-Methyl-2′-deoxycytidine. G at positions 11 and 12 is 2′-O-Methyl-2′-deoxyguanosine. A at position 14 is 2′-O-Methyl-2′-deoxyadenosine. Nucleotide 40 is an inverted orientation T(3′-3′-linked). 108 CACAGGCTAC GGCACGTAGA GCATCACCAT GATCCTGTGT 40 40base pairs nucleic acid single linear DNA unknown C at positions 1 and 3is 2′-O-Methyl- 2′-deoxycytidine. A at positions 2 and 4 is 2′-O-Methyl-2′-deoxyadenosine. Nucleotide 40 is an inverted orientation T(3′-3′-linked). 109 CACAGGCTAC GGCACGTAGA GCATCACCAT GATCCTGTGT 40 40base pairs nucleic acid single linear DNA unknown U at positions 36 and38 is 2′-O- Methyl-2′-deoxyuridine. G at positions 37 and 39 is 2′-O-Methyl-2′-deoxyguanosine. Nucleotide 40 is an inverted orientation T(3′-3′-linked). 110 CACAGGCTAC GGCACGTAGA GCATCACCAT GATCCUGUGT 40 40base pairs nucleic acid single linear unknown C at position 7 is2′-fluoro-2′- deoxycytidine. Nucleotide 40 is an inverted orientation T(3′-3′-linked). 111 CACAGGCTAC GGCACGTAGA GCATCACCAT GATCCTGTGT 40 40base pairs nucleic acid single linear unknown C at position 22 is2′-fluoro-2′- deoxycytidine. Nucleotide 40 is an inverted orientation T(3′-3′-linked). 112 CACAGGCTAC GGCACGTAGA GCATCACCAT GATCCTGTGT 40 40base pairs nucleic acid single linear unknown C at position 25 is2′-fluoro-2′- deoxycytidine. Nucleotide 40 is an inverted orientation T(3′-3′-linked). 113 CACAGGCTAC GGCACGTAGA GCATCACCAT GATCCTGTGT 40 40base pairs nucleic acid single linear unknown C at position 34 is2′-fluoro-2′- deoxycytidine. Nucleotide 40 is an inverted orientation T(3′-3′-linked). 114 CACAGGCTAC GGCACGTAGA GCATCACCAT GATCCTGTGT 40 40base pairs nucleic acid single linear unknown C at position 35 is2′-fluoro-2′- deoxycytidine. Nucleotide 40 is an inverted orientation T(3′-3′-linked). 115 CACAGGCTAC GGCACGTAGA GCATCACCAT GATCCTGTGT 40 40base pairs nucleic acid single linear unknown U at position 8 is2′-fluoro-2′- deoxyuridine. Nucleotide 40 is an inverted orientation T(3′-3′-linked). 116 CACAGGCUAC GGCACGTAGA GCATCACCAT GATCCTGTGT 40 40base pairs nucleic acid single linear unknown U at position 17 is2′-fluoro-2′- deoxyuridine. Nucleotide 40 is an inverted orientation T(3′-3′-linked). 117 CACAGGCTAC GGCACGUAGA GCATCACCAT GATCCTGTGT 40 40base pairs nucleic acid single linear unknown U at position 24 is2′-fluoro-2′- deoxyuridine. Nucleotide 40 is an inverted orientation T(3′-3′-linked). 118 CACAGGCTAC GGCACGTAGA GCAUCACCAT GATCCTGTGT 40 40base pairs nucleic acid single linear unknown U at position 30 is2′-fluoro-2′- deoxyuridine. Nucleotide 40 is an inverted orientation T(3′-3′-linked). 119 CACAGGCTAC GGCACGTAGA GCATCACCAU GATCCTGTGT 40 40base pairs nucleic acid single linear unknown U at position 33 is2′-fluoro-2′- deoxyuridine. Nucleotide 40 is an inverted orientation T(3′-3′-linked). 120 CACAGGCTAC GGCACGTAGA GCATCACCAT GAUCCTGTGT 40 40base pairs nucleic acid single linear unknown C at positions 7, 22, 25,34 and 35 is 2′-fluoro-2′-deoxycytidine. U at positions 8, 17, 24, 33and 36 is 2′-fluoro-2′-deoxyuridine. Nucleotide 40 is an invertedorientation T (3′-3′-linked). 121 CACAGGCUAC GGCACGUAGA GCAUCACCATGAUCCUGTGT 40 40 base pairs nucleic acid single linear unknown C atpositions 10, 13, 27 and 28 is 2′-O-Methyl-2′-deoxycytidine. G atpositions 11, 12, 37 and 39 is 2′-O-Methyl-2′-deoxyguanosine. A atpositions 14, 26 and 29 is 2′-O- Methyl-2′-deoxyadenosine. C at position34 is 2′-fluoro-2′- deoxycytidine. U at position 38 is 2′-O-Methyl-2′-deoxyuridine. Nucleotide 40 is an inverted orientation T (3′-3′-linked).122 CACAGGCTAC GGCACGTAGA GCATCACCAT GATCCTGUGT 40 40 base pairs nucleicacid single linear unknown C at positions 10, 13, 25, 27 and 28 is2′-O-Methyl-2′-deoxycytidine. G at positions 11, 12, 37 and 39 is2′-O-Methyl-2′-deoxyguanosine. A at positions 14, 26 and 29 is 2′-O-Methyl-2′-deoxyadenosine. C at position 34 is 2′-fluoro-2′-deoxycytidine. U at positions 36 and 38 is 2′-O- Methyl-2′-deoxyuridine.Nucleotide 40 is an inverted orientation T (3′-3′-linked). 123CACAGGCTAC GGCACGTAGA GCATCACCAT GATCCUGUGT 40 40 base pairs nucleicacid single linear unknown U at positions 8 and 24 is 2′-fluoro-2′-deoxyuridine. C at positions 10, 13, 27 and 28 is2′-O-Methyl-2′-deoxycytidine. G at positions 11, 12, 37 and 39 is2′-O-Methyl-2′-deoxyguanosine. A at positions 14, 26 and 29 is 2′-O-Methyl-2′-deoxyadenosine. C at positions 25, 34 and 35 is 2′-fluoro-2′-deoxycytidine. U at positions 36 and 38 is 2′-O-Methyl-2′-deoxyuridine. Nucleotide 40 is an inverted orientation T(3′-3′-linked). 124 CACAGGCUAC GGCACGTAGA GCAUCACCAT GATCCUGUGT 40 40base pairs nucleic acid single linear unknown C at positions 10, 13, 25,27 and 28 is 2′-O-Methyl-2′-deoxycytidine. G at positions 11, 12, 37 and39 is 2′-O-Methyl-2′-deoxyguanosine. A at positions 14, 26 and 29 is2′-O- Methyl-2′-deoxyadenosine. C at positions 34 and 35 is 2′-fluoro-2′-deoxycytidine. U at positions 36 and 38 is 2′-O-Methyl-2′-deoxyuridine. Nucleotide 40 is an inverted orientation T(3′-3′-linked). 125 CACAGGCTAC GGCACGTAGA GCATCACCAT GATCCUGUGT 40 40base pairs nucleic acid single linear unknown U at position 8 is2′-fluoro-2′- deoxyuridine. C at positions 10, 13, 25, 27 and 28 is2′-O-Methyl-2′-deoxycytidine. G at positions 11, 12, 37 and 39 is2′-O-Methyl-2′-deoxyguanosine. A at positions 14, 26 and 29 is 2′-O-Methyl-2′-deoxyadenosine. C at position 34 is 2′-fluoro-2′-deoxycytidine. U at positions 36 and 38 is 2′-O- Methyl-2′-deoxyuridine.Nucleotide 40 is an inverted orientation T (3′-3′-linked). 126CACAGGCUAC GGCACGTAGA GCATCACCAT GATCCUGUGT 40 36 base pairs nucleicacid single linear unknown U at positions 6 and 22 is 2′-fluoro-2′-deoxyuridine. C at positions 8, 11, 25 and 26 is 2′-O-Methyl-2′-deoxycytidine. G at positions 9, 10, and 35 is 2′-O-Methyl-2′-deoxyguanosine. A at positions 12, 24 and 27 is 2′-O-Methyl-2′-deoxyadenosine. C at positions 23, 32 and 33 is 2′-fluoro-2′-deoxycytidine. U at position 34 is 2′-O-Methyl-2′-deoxyuridine. Nucleotide 36 is an inverted orientation T (3′-3′-linked).127 CAGGCUACGG CACGTAGAGC AUCACCATGA TCCUGT 36 34 base pairs nucleicacid single linear unknown U at positions 6 and 20 is 2′-fluoro-2′-deoxyuridine. C at positions 7, 10, 23 and 24 is 2′-O-Methyl-2′-deoxycytidine. G at positions 8, 9, and 33 is 2′-O-Methyl-2′-deoxyguanosine. A at positions 11, 22 and 25 is 2′-O-Methyl-2′-deoxyadenosine. C at positions 21, 30 and 31 is 2′-fluoro-2′-deoxycytidine. U at position 32 is 2′-O-Methyl-2′-deoxyuridine. Nucleotide 34 is an inverted orientation T (3′-3′-linked).128 CAGGCUCGGC ACGAGAGCAU CACCATGATC CUGT 34 32 base pairs nucleic acidsingle linear unknown U at positions 6 and 20 is 2′-fluoro-2′-deoxyuridine. C at positions 7, 10, 22 and 23 is 2′-O-Methyl-2′-deoxycytidine. G at positions 8, 9, and 31 is 2′-O-Methyl-2′-deoxyguanosine. A at positions 11 and 24 is 2′-O-Methyl-2′-deoxyadenosine. C at positions 21, 28 and 29 is 2′-fluoro-2′-deoxycytidine. U at position 30 is 2′-O-Methyl-2′-deoxyuridine. Nucleotide 32 is an inverted orientation T (3′-3′-linked).129 CAGGCUCGGC ACGAGAGCAU CCCAGATCCU GT 32 36 base pairs nucleic acidsingle linear unknown C at positions 8, 11, 23, 25, 26, 32 and 33 is2′-O-Methyl-2′-deoxycytidine. G at positions 9, 10, and 35 is 2′-O-Methyl-2′-deoxyguanosine. A at positions 12, 24 and 27 is 2′-O-Methyl-2′-deoxyadenosine. U at position 6 is 2′-fluoro-2′- deoxyuridine.U at positions 22 and 34 is 2′-O- Methyl-2′-deoxyuridine. Nucleotide 36is an inverted orientation T (3′-3′-linked). 130 CAGGCUACGG CACGTAGAGCAUCACCATGA TCCUGT 36 36 base pairs nucleic acid single linear unknown Cat positions 5, 8, 11, 23, 25, 26, 32 and 33 is2′-O-Methyl-2′-deoxycytidine. (ix) FEATURE (D) OTHER INFORMATION U atpositions 6, 22 and 34 is 2′-O- Methyl-2′-deoxyuridine. A at positions7, 12, 24 and 27 is 2′- O-Methyl-2′-deoxyadenosine. G at positions 9,10, and 35 is 2′-O- Methyl-2′-deoxyguanosine. Nucleotide 36 is aninverted orientation T (3′-3′-linked). 131 CAGGCUACGG CACGTAGAGCAUCACCATGA TCCUGT 36 36 base pairs nucleic acid single linear unknown Cat positions 5, 8, 11, 23, 25, 26, 32 and 33 is2′-O-Methyl-2′-deoxycytidine. U at positions 6, 22 and 34 is 2′-O-Methyl-2′-deoxyuridine. A at positions 12, 18, 24 and 27 is 2′-O-Methyl-2′-deoxyadenosine. G at positions 9, 10, and 35 is 2′-O-Methyl-2′-deoxyguanosine. Nucleotide 36 is an inverted orientation T(3′-3′-linked). 132 CAGGCUACGG CACGTAGAGC AUCACCATGA TCCUGT 36 36 basepairs nucleic acid single linear unknown C at positions 5, 8, 11, 23,25, 26, 32 and 33 is 2′-O-Methyl-2′-deoxycytidine. U at positions 6, 22and 34 is 2′-O- Methyl-2′-deoxyuridine. G at positions 9, 10, 19 and 35is 2′- O-Methyl-2′-deoxyguanosine. A at positions 12, 24 and 27 is 2′-O-Methyl-2′-deoxyadenosine. Nucleotide 36 is an inverted orientation T(3′-3′-linked). 133 CAGGCUACGG CACGTAGAGC AUCACCATGA TCCUGT 36 36 basepairs nucleic acid single linear unknown U at positions 6 and 22 is2′-fluoro- 2′-deoxyuridine. C at positions 8, 11, 25 and 26 is 2′-O-Methyl-2′-deoxycytidine. G at positions 9, 10, 19 and 35 is 2′-O-Methyl-2′-deoxyguanosine. A at positions 12, 24 and 27 is 2′-O-Methyl-2′-deoxyadenosine. C at positions 23, 32 and 33 is 2′-fluoro-2′-deoxycytidine. U at position 34 is 2′-O-Methyl-2′-deoxyuridine. Nucleotide 36 is an inverted orientation T (3′-3′-linked).134 CAGGCUACGG CACGTAGAGC AUCACCATGA TCCUGT 36 36 base pairs nucleicacid single linear unknown U at positions 6 and 22 is 2′-fluoro-2′-deoxyuridine. C at positions 8, 11, 20, 25 and 26 is2′-O-Methyl-2′-deoxycytidine. G at positions 9, 10, 19 and 35 is 2′-O-Methyl-2′-deoxyguanosine. A at positions 12, 24 and 27 is 2′-O-Methyl-2′-deoxyadenosine. C at positions 23, 32 and 33 is 2′-fluoro-2′-deoxycytidine. U at position 34 is 2′-O-Methyl-2′-deoxyuridine. Nucleotide 36 is an inverted orientation T (3′-3′-linked).135 CAGGCUACGG CACGTAGAGC AUCACCATGA TCCUGT 36 36 base pairs nucleicacid single linear unknown U at positions 6 and 22 is 2′-fluoro- 2′-deoxyuridine. C at positions 8, 11, 20, 25 and 26 is2′-O-Methyl-2′-deoxycytidine. G at positions 9, 10, 17, 19 and 35 is2′-O-Methyl-2′-deoxyguanosine. A at positions 12, 24 and 27 is 2′-O-Methyl-2′-deoxyadenosine. C at positions 23, 32 and 33 is 2′-fluoro-2′-deoxycytidine. U at position 34 is 2′-O-Methyl-2′-deoxyuridine. Nucleotide 36 is an inverted orientation T (3′-3′-linked).136 CAGGCUACGG CACGTAGAGC AUCACCATGA TCCUGT 36 36 base pairs nucleicacid single linear unknown U at positions 6 and 22 is 2′-fluoro-2′-deoxyuridine. C at positions 8, 11, 25 and 26 is 2′-O-Methyl-2′-deoxycytidine. G at positions 9, 10, 17, 19 and 35 is2′-O-Methyl-2′-deoxyguanosine. A at positions 12, 24 and 27 is 2′-O-Methyl-2′-deoxyadenosine. C at positions 23, 32 and 33 is 2′-fluoro-2′-deoxycytidine. U at position 34 is 2′-O-Methyl-2′-deoxyuridine. Nucleotide 36 is an inverted orientation T (3′-3′-linked).137 CAGGCUACGG CACGTAGAGC AUCACCATGA TCCUGT 36 36 base pairs nucleicacid single linear unknown U at positions 6 and 22 is 2′-fluoro-2′-deoxyuridine. A at positions 7, 12, 24 and 27 is 2′-O-Methyl-2′-deoxyadenosine. C at positions 8, 11, 25 and 26 is 2′-O-Methyl-2′-deoxycytidine. G at positions 9, 10, 17, 19 and 35 is2′-O-Methyl-2′-deoxyguanosine. C at positions 23, 32 and 33 is 2′-fluoro-2′-deoxycytidine. U at position 34 is 2′-O-Methyl-2′-deoxyuridine. Nucleotide 36 is an inverted orientation T (3′-3′-linked).138 CAGGCUACGG CACGTAGAGC AUCACCATGA TCCUGT 36 32 base pairs nucleicacid single linear unknown U at positions 6 and 20 is 2′-fluoro-2′-deoxyuridine. C at position 8 is 2′-O-Methyl-2′- deoxycytidine. G atpositions 9, 17, and 31 is 2′-O- Methyl-2′-deoxyguanosine. S atpositions 10 and 23 is a hexaethyleneglycol spacer. C at positions 21,28 and 29 is 2′- fluoro-2′-deoxycytidine. A at position 22 is2′-O-Methyl-2′- deoxyadenosine. U at position 30 is 2′-O-Methyl-2′-deoxyuridine. Nucleotide 32 is an inverted orientation T (3′-3′-linked).139 CAGGCUACGS CGTAGAGCAU CASTGATCCU GT 32 32 base pairs nucleic acidsingle linear unknown U at positions 6 and 20 is 2′-fluoro-2′-deoxyuridine. C at position 8 is 2′-O-Methyl-2′- deoxycytidine. G atpositions 9, 15, 17, and 31 is 2′-O-Methyl-2′-deoxyguanosine. S atpositions 10 and 23 is a hexaethyleneglycol spacer. C at positions 21,28 and 29 is 2′- fluoro-2′-deoxycytidine. A at position 22 is2′-O-Methyl-2′- deoxyadenosine. U at position 30 is 2′-O-Methyl-2′-deoxyuridine. Nucleotide 32 is an inverted orientation T (3′-3′-linked).140 CAGGCUACGS CGTAGAGCAU CASTGATCCU GT 32 36 base pairs nucleic acidsingle linear unknown Nucleotide 36 is an inverted orientation T(3′-3′-linked). 141 CAGGCTACGG CACGTAGAGC ATCACCATGA TCCTGT 36 32 basepairs nucleic acid single linear unknown U at positions 6, 20 and 30 is2′- fluoro-2′-deoxyuridine. C at positions 8, 21, 28 and 29 is 2′-fluoro-2′-deoxycytidine. G at positions 9, 15, 17, and 31 is 2′-O-Methyl-2′-deoxyguanosine. S at positions 10 and 23 is ahexaethyleneglycol spacer. A at position 22 is 2′-O-Methyl-2′-deoxyadenosine. Nucleotide 32 is an inverted orientation T(3′-3′-linked). 142 CAGGCUACGS CGTAGAGCAU CASTGATCCU GT 32 32 base pairsnucleic acid single linear unknown U at positions 6, 20 and 30 is 2′-fluoro-2′-deoxyuridine. C at positions 8, 21, 28 and 29 is 2′-fluoro-2′-deoxycytidine. G at positions 9, 15, 17, and 31 is 2′-O-Methyl-2′-deoxyguanosine. N at positions 10 and 23 is ahexaethyleneglycol spacer. A at position 22 is 2′-O-Methyl-2′-deoxyadenosine. Nucleotide 32 is an inverted orientation T(3′-3′-linked). 143 CAGGCUACGN CGTAGAGCAU CANTGATCCU GT 32 32 base pairsnucleic acid single linear unknown G at positions 3, 4, 12, and 25 is2′-O-Methyl-2′-deoxyguanosine. U at positions 6, 20 and 27 is 2′-fluoro-2′-deoxyuridine. N at positions 10 and 23 is a hexaethyleneglycolspacer. C at positions 11, 18, 21 and 29 is 2′-fluoro-2′-deoxycytidine.A at position 16 is 2′-O-Methyl-2′- deoxyadenosine. Nucleotide 32 is aninverted orientation T (3′-3′-linked). 144 CAGGCUACGN CGTAGAGCAUCANTGAUCCT GT 32 32 base pairs nucleic acid single linear unknown C atpositions 4, 8, 21 and 29 is 2′- fluoro-2′-deoxycytidine. U at positions6, 20 and 30 is 2′- fluoro-2′-deoxyuridine. G at positions 5, 9, 17, and31 is 2′-O-Methyl-2′-deoxyguanosine. A at position 22 is 2′-O-Methyl-2′-deoxyadenosine. N at positions 10 and 23 is a hexaethylene glycolphosphoramidite. Nucleotide 32 is an inverted orientation T(3′-3′-linked). 145 CAGCGUACGN CGTACCGATU CANTGAAGCU GT 32 32 base pairsnucleic acid single linear unknown U at positions 6, 20 and 30 is 2′-fluoro-2′-deoxyuridine. C at positions 8, 21, 28, and 29 is2′-fluoro-2′-deoxycytidine. G at positions 9, 15, 17, and 31 is2′-O-Methyl-2′-deoxyguanosine. A at position 22 is 2′-O-Methyl-2′-deoxyadenosine. N at positions 10 and 23 is a hexaethylene glycolphosphoramidite. Nucleotide 32 is an inverted orientation T(3′-3′-linked). 146 CAGGCUACGN CGTAGAGCAU CANTGATCCU GT 32 32 base pairsnucleic acid single linear unknown C at positions 4, 8, 21 and 29 is 2′-fluoro-2′-deoxycytidine. U at positions 6, 20 and 30 is 2′-fluoro-2′-deoxyuridine. G at positions 5, 9, 17, and 31 is2′-O-Methyl-2′-deoxyguanosine. A at position 22 is 2′-O-Methyl-2′-deoxyadenosine. N at positions 10 and 23 is a hexaethylene glycolphosphoramidite. Nucleotide 32 is an inverted orientation T(3′-3′-linked). 147 CAGCGUACGN CGTACCGATU CANTGAAGCU GT 32 32 base pairsnucleic acid single linear unknown U at positions 6, 20 and 30 is 2′-fluoro-2′-deoxyuridine. C at positions 8, 21, 28 and 29 is 2′-fluoro-2′-deoxycytidine. G at positions 9, 15, 17, and 31 is2′-O-Methyl-2′-deoxyguanosine. S at positions 10 and 23 is ahexaethyleneglycol spacer. A at position 22 is 2′-O-Methyl-2′-deoxyadenosine. Nucleotide 32 is an inverted orientation T(3′-3′-linked). 148 CAGGCUACGS CGTAGAGCAU CASTGATCCU GT 32 32 base pairsnucleic acid single linear unknown C at positions 4, 8, 21 and 29 is 2′-fluoro-2′-deoxycytidine. G at positions 5, 9, 17, and 31 is 2′-O-Methyl-2′-deoxyguanosine. U at positions 6, 20 and 30 is 2′-fluoro-2′-deoxyuridine. N at positions 10 and 23 is a hexaethyleneglycolspacer. A at position 22 is 2′-O-Methyl-2′- deoxyadenosine. Nucleotide32 is an inverted orientation T (3′-3′-linked). 149 CAGCGUACGNCGTACCGATU CANTGAAGCU GT 32

We claim:
 1. A Complex comprised of a platelet-derived growth factor(PDGF) Nucleic Acid Ligand and a Non-Immunogenic, High Molecular WeightCompound.
 2. The Complex of claim 1 further comprising a Linker betweensaid Ligand and said Non-Immunogenic, High Molecular Weight Compound. 3.The Complex of claim 1 wherein said Ligand comprises a Linker.
 4. TheComplex of claim 1 wherein said Non-Immunogenic, High Molecular WeightCompound is a Polyalkylene Glycol.
 5. The Complex of claim 4 whereinsaid Polyalkylene Glycol is polyethylene glycol.
 6. The Complex of claim5 wherein said polyethylene glycol has a molecular weight of about 10-80K.
 7. The Complex of claim 6 wherein said polyethylene glycol has amolecular weight of about 20-45 K.
 8. A Lipid Construct comprising theComplex of claim
 1. 9. The Complex of claim 1 wherein said PDGF NucleicAcid Ligand is identified from a Candidate Mixture of Nucleic Acidsaccording to the method comprising: a) contacting the Candidate Mixturewith PDGF, wherein the Nucleic Acids having an increased affinity toPDGF relative to the Candidate Mixture may be partitioned from theremainder of the Candidate Mixture; b) partitioning the increasedaffinity Nucleic Acids from the remainder of the Candidate Mixture; andc) amplifying the increased affinity Nucleic Acids to yield a mixture ofNucleic Acids enriched for Nucleic Acids having an increased affinityfor PDGF; whereby a Nucleic Acid Ligand of PDGF is identified.
 10. TheComplex of claim 9 wherein the method further comprises repeating stepsb) and c).
 11. The complex of claim 7 wherein said Complex is

in which said PEG spacer is

and said nucleic acid ligand comprises SEQ ID NO:146.